Peptide coacervates and methods of use thereof

ABSTRACT

The present invention provides for a composition, as disclosed herein, for delivery of an active agent. The composition includes a peptide coacervate, wherein the peptide coacervate includes one or more peptides derived from histidine-rich proteins, and an active agent encapsulated in the peptide coacervate. Further provided are a method for encapsulation of an active agent in a peptide coacervate, a method for delivery of an active agent, and a method for treating or diagnosing a condition or disease in a subject in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to Singapore Application No.10201805304Y entitled “Peptide Coacervates For Encapsulation of InsulinWith High Efficiency And Glucose-Responsive Release”, filed on Jun. 20,2018, the disclosures of which is incorporated herein by reference inits entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 690148_558D1_SEQUENCE_LISTING.txt. The text fileis 29 KB, was created on Oct. 20, 2021, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present invention lies in the field of targeted delivery of activeagents using peptide coacervates and methods of coacervate formation andactive agent encapsulation.

BACKGROUND

The Humboldt squid beak includes a hard biomolecular composite made ofchitin and proteins. The squid beak proteins were recently isolated andsequenced and two families of proteins, chitin binding beak proteins(DgCBPs) and histidine-rich beak proteins (DgHBPs) were discoveredwithin the beak. DgCBPs likely bind to chitin to form a chitin-DgCBPsscaffold, while DgHBPs exhibit self-coacervation ability, aliquid-liquid phase separation (LLPS) process resulting in the formationof highly concentrated protein microdroplets. DgHBP coacervates havebeen hypothesized to infiltrate the chitin-DgCBPs scaffold (Tan et al.(2015) Nat. Chem. Biol. 11 (7), 488-95) followed by interchain covalentcross-linking during maturation, with the very high cross-link densityimparting the beak with its impressive mechanical properties (Miserez etal. (2007) Acta Biomater. 3 (1), 139-49; Miserez et al. (2010) J. Biol.Chem. 285 (49), 38115-24). The DgHBPs identified have been sequenced andhave been found to exhibit a two-domain organisation. The N-terminaldomains contain non-repetitive, long stretches of Alanine (Ala) andHistidine (His)-rich regions, whereas the C-terminal domains includestandem His- and Gly-rich penta-repeats (GAGFA, GHGXX′/X″ or GHGXY, whereX represents a hydrophobic residue, X′ usually represents tyrosine andX″ represents either glycine or alanine. The C-terminal domain motifswere found to be responsible for DgHBPs self-coacervation properties(Cai et al. (2017) Soft Matter 13 (42), 7740-7752).

Coacervation or LLPS is the demixing of a homogeneous polymer solutioninto two distinct phases: a concentrated macromolecule-rich (orcoacervate) phase and a dilute macromolecule-depleted phase. Whilecoacervation studies were initiated in the field of biopolymericcolloids, in recent years LLPS has attracted considerable interest fromlife scientists, with numerous studies showing its role in organizingbiomolecules in living cells via formation of membrane-less organelles.Another less recognized but increasingly appreciated biological role ofLLPS is associated with the assembly of extracellular, load-bearingstructures (Muiznieks et al. (2018) J. Mol. Biol.doi:10.1016/J.JMB.2018.06.010). A well-known example is tropoelastin,which undergoes self-coacervation upon secretion into the extracellularmatrix where it self-assembles to form elastic fibers that providestrength and resilience to elastic tissues (Yeo et al. (2011) Adv.Colloid Interface Sci. 167, 94-103). Coacervation has also beenrecognized to play a key role in natural bioadhesives secreted by marineinvertebrates, for example the sandcastle tubeworm or mussels (Wei etal. (2014) Acta Biomater. 10, 1663-1670) and to be involved in theformation of biological composite materials.

Recent studies of proteins involved in LLPS have revealed that theyusually belong to the family of intrinsically disordered proteins (IDPs)or contain intrinsically disordered regions (IDRs). Suchproteins/regions are characterized by very dynamic molecularconformations and a low sequence complexity with a modular organizationof their primary structure (Brangwynne, et al. (2015) Nat. Phys. 11,899-904; Uversky (2017) Adv. Colloid Interface Sci. 239, 97-114; Van DerLee et al. (2014) Chem. Rev. 114, 6589-6631). As a result, they lack thewell-defined three-dimensional structure typical of globular proteins.

It has been suggested that various intra- or intermolecular interactionsare involved during LLPS of IDPs/IDRs, for example multivalent(cooperative), electrostatic, hydrophobic, or cation-π interactions.Structure-function relationships of IDPs have primarily been obtained bysite-directed mutagenesis, establishing the contributions of individualresidues to the phase separation process. However, molecular-scaleinteractions behind LLPS are still sparsely understood.

While in various fields of technology, in particular in pharmaceutical,diagnostic, chemical and biological applications, a multitude ofdelivery methods are known, there is still need for novel and improveddelivery systems, in particular those suited to deliver pharmaceuticallyactive agents to a patient in need thereof, including release of thepharmaceutically active agents at a specific site at a specific time orover a specific time period or if a specific condition is met.

Such delivery systems would, for example, be useful in the treatment andmanagement of diabetes. Diabetes is a chronic metabolic disease that ischaracterized by abnormal high levels of fasting blood glucose. As ageneral practice, insulin is often administrated to control the bloodglucose level in both type I and II diabetic patients. To obtain idealtherapeutic results, a strict insulin administration program is neededfor diabetic patients. Hence, many investigations have been dedicated todevelop environment-responsive delivery systems. Commonly, aglucose-responsive insulin delivery system (GRIDS) is able to sense theincreased glucose concentration and subsequently release the requiredamount of insulin according to the glucose level (Farmer et al. (2008)J. Pharm. Pharmacol. 60 (1), 1-13; Thabit and Hovorka (2014) Curr. Opin.Endocrinol. Diabetes Obes. 21 (2), 95-101; Battelino et al. (2015) BestPract Res. Clin Endocrinol Metab 29 (3), 315-25; Xie et al. (2017)Macromol. Rapid Commun. 38 (23), 1700413).

GRIDS may not only replace the recurrent insulin injections, but canalso reduce the patient's direct involvement in glucose control andprevent insulin from excessive or insufficient dosage (Yaturu (2013)World J. Diabetes 4 (1), 1-7; Shah et al. (2016) Int. J. Pharm. Investig6 (1), 1-9; Webber and Anderson (2015) J. Drug Target 23 (7-8), 651-5).One approach used for sensing glucose is to incorporate aglucose-responsive element such as glucose oxidase (GOx) into thedelivery system, whereby GOx catalyzes the conversion of D-glucose intogluconic acid to reduce the local pH (Webber and Anderson, supra; Li etal. (2017) Acta Biomater. 51, 294-303; Tai et al. (2014)Biomacromolecules 15 (10), 3495-502; Zhao et al. (2017) Polymers 9 (7),255). The acidic pH subsequently results in the conformational orstructural changes of the carrier and ultimately releases the insulin.Such a strategy has been notably employed in pH-responsive hydrogels(Zhao et al., supra).

Diabetes management is thus one example of a field where there is stillparticular need for efficient and controllable delivery systems.

SUMMARY

Peptide coacervates formed from peptides derived from histidine-richproteins can be used for the efficient delivery of active agents. Thepeptide coacervates formed may co-encapsulate two or more active agentsto be applicable and effective in the management and/or treatment ofdiseases or disorders, such as cancer or diabetes. Additionally, theinventors' findings provide general guidelines and concepts fordesigning (pH-responsive) peptides with liquid-liquid phase separation(LLPS) ability for various applications, including bio-inspiredprotocells and smart drug-delivery systems.

In a first aspect, a composition for delivery of an active agent mayinclude a peptide coacervate, said peptide coacervate comprisingpeptides derived from histidine-rich proteins, and said active agent,wherein the active agent is encapsulated in the coacervate.

In various embodiments, the composition is an aqueous liquid two phaseformulation, comprising (a) a coacervate colloidal phase comprising thepeptides derived from histidine-rich proteins and the active agent; and(b) a dilute aqueous phase. In some embodiments, the colloidal phase hasthe form of droplets having substantially spherical shape with adiameter ranging from about 0.2 to about 5 μm.

In various non-limiting embodiments, the histidine-rich proteins arehistidine-rich beak proteins (DgHBPs), for example derived from the beakof a squid, in particular the Humbold squid (Dosidicus gigas). Thehistidine-rich proteins may, for example, include, but not limited to,DgHBP-1 (SEQ ID NO:1) and DgHBP-2 (SEQ ID NO:2), with the peptidesderived therefrom being, in various embodiments, fragments thereof thatcomprise at least one copy of the peptide motif GHGXY (SEQ ID NO:25),wherein X is valine (V), leucine (L) or proline (P), such as GHGLY (SEQID NO:46). Further motifs that may be comprised include, but are notlimited to GHGLX (SEQ ID NO:26), wherein X is leucine (L), histidine(H), tyrosine (Y) or glycine (G), such as GHGLH (SEQ ID NO:28), andGAGFA (SEQ ID NO:27) and GFA.

In various embodiments, the peptides derived from histidine-richproteins comprise the amino acid sequence

(GHGX¹Y)_(a)[(GX²GX³A)_(b)(GHGLX⁴)_(c)(GFA)_(d)]_(f)(GHGX¹Y)_(a)

wherein X¹ is valine (V), leucine (L) or proline (P), X² is alanine (A)or proline (P); X³ is phenylalanine (F) or tyrosine (Y), X⁴ is leucine(L), histidine (H), tyrosine (Y) or glycine (G),

each a is 0 or an integer≥1;

each b is 0 or an integer≥1;

each c is 0 or an integer≥1;

each d is 0 or an integer≥1;

f is an integer≥1;

with the sum of all a being ≥2 and the sum of all a+b+c+d is ≥4.

In non-limiting embodiments, the peptides derived from histidine-richproteins may include an amino acid sequence, such as but not limited to:

-   -   (1) (GHGXY)_(n),    -   wherein X is valine (V), leucine (L) or proline (P), and n is        ≥4;    -   (2) [(GHGXY)_(n)(GAGFA)_(m)]_(i)GHGXY    -   wherein X is valine (V), leucine (L) or proline (P); n is 1; m        is ≥1; and i≥2;    -   (3) [(GHGXY)_(n)(GHGLH)_(m)]_(i)GHGXY    -   wherein X is valine (V), leucine (L) or proline (P); n is ≥1; m        is ≥1; and i≥2;    -   (4) (GHGXY)_(n)(GAGFA)_(m)GHGXY    -   wherein X is valine (V), leucine (L) or proline (P); n is ≥1;        and m is ≥2; and    -   (5) (GHGXY)_(n)(GHGLH)_(m)GHGXY    -   wherein X is valine (V), leucine (L) or proline (P); n is ≥1;        and m is ≥2; and    -   (6) combinations of the above.

Non-limiting peptides may include an amino acid sequence, such as butnot limited to,

(SEQ ID NO: 3) (i) GHGXY GHGXY GHGXY GHGXY GHGXY W  (SEQ ID NO: 4)(ii) GHGXY GHGXY GHGXY GHGXY GHGXY  (SEQ ID NO: 5)(iii) GHGXY GAGFA GHGXY GAGFA GHGXY  (SEQ ID NO: 6)(iv) GHGXY GHGLH GHGLH GHGLH GHGXY  (SEQ ID NO: 7)(v) GHGXY GAGFA GAGFA GAGFA GHGXY  (SEQ ID NO: 8)(vi) GHGXY GHGXY GHGXY GHGXY  (SEQ ID NO: 9)(vii) GHGXY GAGFA GHGLH GFA GHGXY  (SEQ ID NO: 10)(viii) GHGXY GAGFA GHGLH GAGFA GHGXY (SEQ ID NO: 11)(ix) GHGXY GHGLH GAGFA GHGLH GHGXY  (SEQ ID NO: 12)(x) GHGXY GAGFA GAGFA GHGLH GHGXY  (SEQ ID NO: 13)(xi) GHGXY GHGLH GAGFA GAGFA GHGXY  (SEQ ID NO: 14)(xii) GHGVY GHGVY GHGPY GHGPY GHGLY W (SEQ ID NO: 15)(xiii) GHGVY GHGVY GHGPY GHGPY GHGLY  (SEQ ID NO: 16)(xiv) GHGLY GAGFA GHGLY GAGFA GHGLY  (SEQ ID NO: 17)(xv) GHGLY GHGLH GHGLH GHGLH GHGLY  (SEQ ID NO: 18)(xvi) GHGLY GAGFA GAGFA GAGFA GHGLY  (SEQ ID NO: 19)(xvii) GHGLY GHGLY GHGLY GHGLY  (SEQ ID NO: 20)(xviii) GHGLY GAGFA GHGLH GFA GHGLY  (SEQ ID NO: 21)(xix) GHGLY GAGFA GHGLH GAGFA GHGLY  (SEQ ID NO: 22)(xx) GHGLY GHGLH GAGFA GHGLH GHGLY  (SEQ ID NO: 23)(xxi) GHGLY GAGFA GAGFA GHGLH GHGLY  (SEQ ID NO: 24)(xxii) GHGLY GHGLH GAGFA GAGFA GHGLY,  and (xxiii) combinations thereof 

The active agent may be or include, but is not limited to, proteins,(poly)peptides, carbohydrates, nucleic acids, lipids, chemical compoundsand nanoparticles. Suitable nanoparticles may be or include, but notlimited to, metal nanoparticles, metal oxide nanoparticles, andcombinations thereof. The nanoparticles may be magnetic nanoparticles.

In various embodiments, the active agent is a pharmaceutical ordiagnostic agent. The active agent may comprise or be insulin eitheralone or in combination with the enzyme glucose oxidase.

In various other embodiments, the pharmaceutical or diagnostic agentcomprises or is an anti-cancer agent, such as doxorubicin either aloneor in combination with magnetic nanoparticles.

The composition may be a pharmaceutical or diagnostic formulation foradministration to a subject. In various embodiments it can thus compriseany one or more auxiliaries, carriers and excipients that arepharmaceutically or diagnostically acceptable. In some embodiments, thecomposition is a liquid. The subject may be a mammal, for example ahuman being.

The peptide coacervate may be covalently crosslinked. The crosslinkingmay be achieved by use of catechol moieties, for example by a redoxreaction. In one embodiment, the peptide coacervate is crosslinked with4-methylcatechol (4-MC) and sodium periodate (NaIO₄).

The pH of the composition is, in various embodiments, 7.0 or higher, forexample in the range of 7.4 to 9.5.

A method for the encapsulation of an active agent in a peptidecoacervate may include:

(a) providing an aqueous solution of coacervate-forming peptides,wherein said peptides are derived from histidine-rich proteins; and

(b) combining the aqueous solution of coacervate-forming peptides withthe active agent to induce coacervate formation.

In these methods, the pH of the aqueous solution of thecoacervate-forming peptides may be below 7, for example below 6.5 orbelow 6.0 or even below 5.5.

In various embodiments, the active agents in the combined aqueoussolution are also provided in form of an aqueous solution. Said aqueoussolution may have a pH>7 and, in some embodiments, is buffered such thatthe combination of the aqueous solution of the active agent with theaqueous solution of the coacervate-forming peptides obtained in thecombined aqueous solution has a pH>7. In various embodiments, thecombination of the aqueous solution with the active agent changes the pHof the solution in which the peptide for coacervate forming is solved togreater than 7 and thus initiates coacervate formation.

In various embodiments, the concentration of the coacervate-formingpeptides in the provided aqueous solution is greater than about 0.3mg/mL and may, for example, range from about 0.3 to about 100 mg/mL.

The combined solution after coacervate formation may be an aqueousliquid two phase formulation, comprising (1) a coacervate colloidalphase comprising the peptides derived from histidine-rich proteins andthe active agent; and (2) a dilute aqueous phase.

The coacervates formed may have the form of droplets havingsubstantially spherical shape with a diameter ranging from about 0.2 toabout 5 μm.

In various non-limiting embodiments of the methods, the histidine-richproteins and peptides are as defined above for the inventivecompositions.

In still another non-limiting aspect, a method for the delivery of anactive agent may include:

(i) providing a composition comprising a peptide coacervate, saidpeptide coacervate comprising peptides derived from histidine-richproteins, and said active agent, wherein the active agent isencapsulated in the coacervate;

(ii) releasing said active agent from the coacervates by exposing thecoacervates to conditions that trigger the release of the active agent.

In various embodiments of the above methods, the conditions that triggerthe release of the active agent may be or include, but not limited to,elevated temperatures, pH changes, exposure to release agents andcombinations thereof.

In a still further non-limiting aspect, a method for treating ordiagnosing a condition or disease in a subject in need thereof mayinclude:

(i) administering a composition comprising a peptide coacervate, saidpeptide coacervate comprising peptides derived from histidine-richproteins, and a pharmaceutical or diagnostic agent, wherein thepharmaceutical or diagnostic agent is encapsulated in the coacervate tosaid subject;

(ii) facilitating the release of said pharmaceutical or diagnostic agentfrom the coacervate by exposing the coacervate to conditions thattrigger the release of the pharmaceutical or diagnostic agent Theconditions that trigger the release of the pharmaceutical or diagnosticagent may be selected from those disclosed above for the deliverymethods. The subject may be a mammal, for example a human.

In non-limiting embodiments, the subject is a human afflicted bydiabetes, wherein the pharmaceutical or diagnostic agent is insulin,wherein the coacervate further comprises encapsulated glucose oxidase,and wherein release is facilitated by an increase in glucoseconcentration and the resulting acidification of the coacervate.

In further exemplary embodiments, the subject is a human afflicted bycancer, wherein the pharmaceutical or diagnostic agent is doxorubicin,wherein the coacervate further comprises encapsulated magneticnanoparticles, and wherein release is facilitated by exposure of thesubject to a magnetic field resulting in a temperature increase in thecoacervate.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Schematic representation of the glucose-responsive insulindelivery system (GRIDS) based on DgHBP-2 derived peptide coacervates.(A) Illustration of the coacervation and encapsulation process. (B) Whenthe coacervate droplets are exposed to glucose, the latter diffuses intothe droplets and is converted into gluconic acid. This results in alocal decrease of pH and in turn to the dissociation of coacervatedroplets with the concomitant release of insulin.

FIGS. 2A-2B. Coacervation of DgHBP-2 peptide. (A) Sequence of DgHBP-2derived peptide (SEQ ID NO:14). (B) Relative turbidity of DgHBP-2derived peptide at different pH and Ionic strength (n=3, mean values±S.D.). (C) Microscopy image of DgHBP-2 derived peptide coacervates atpH 7.5, 0.1 M ionic strength.

FIGS. 3A-3B. Dynamic light scattering of (A) DgHBP-2 derived peptidecoacervates and (B) GOx+insulin loaded coacervates in phosphate buffer.A slight increase in hydrodynamic radius of ˜6% (from 1000 to 1060 nm)was observed after encapsulation.

FIG. 4. Microscopy images of DgHBP-2 derived peptide coacervates (1mg/mL) loaded with FITC-insulin (0.1 mg/mL). Light microscopy image(left); fluorescence image (middle); merged microscopy image (right).The grey droplets and green fluorescence are DgHBP-2 derived peptidecoacervates and FITC-insulin, respectively.

FIGS. 5A-5B. Relative cell viability of (A) DgHBP-2 derived peptide, and(B) Insulin-loaded coacervates. Control cells were normalized at 100%(n=6, mean±S.D.).

FIG. 6. Encapsulation efficiency of different concentration ofFITC-insulin by DgHBP-2 derived peptide coacervates (n=3, mean values±S.D.).

FIG. 7. Encapsulation efficiency of FITC-insulin by differentconcentration of DgHBP-2 derived peptide coacervates (n=3, mean values±S.D.).

FIGS. 8A-8D RP-HPLC absorbance chromatograms of (A) DgHBP-2 derivedpeptide (1 mg/mL); (B) native insulin (0.1 mg/mL); (C) supernatant aftercoacervation; and (D) dissolved coacervates pellet after encapsulation.Black arrow indicates the retention time for native insulin. Afterencapsulation, almost no insulin was detected in the supernatant,whereas by dissolving the coacervates after encapsulation, insulin wasfound within the coacervates. These data confirm the high encapsulationefficiency of insulin within the coacervates as measured forFITC-labeled insulin by fluorescence microscopy (FIG. 9A).

FIGS. 9A-9D. Microscopy images of DgHBP-2 derived peptide coacervates (1mg/mL) loaded with FITC-insulin (0.1 mg/mL) and RITC-GOx (0.1 mg/mL).(A) Green fluorescence micrograph; (B) red fluorescence micrograph; (C)light microscopy micrograph; (D) merged (green, red, and light)micrograph image. Yellow fluorescence of the droplets indicateco-encapsulation of FITC-insulin and RITC-GOx.

FIGS. 10A-10B In vitro release assay of insulin from coacervates (A)GOx+insulin with 4 mg/mL of glucose (square, black); GOx+insulin(triangle, blue); Insulin (inverted triangle, pink) and Insulin with 4mg/mL of glucose (circle, red). (B) Alternate high glucose (4 mg/mL,blue) and low glucose (1 mg/mL, green) solution (n=3, mean values±S.D.).

FIGS. 11A-11B. Comparison of released insulin from dissociatedcoacervates and native insulin. (A) CD spectra of regular insulin,denatured insulin, insulin released from dissociated coacervates, andDgHBP-2 peptide. (B) Enzyme-linked immunosorbent assay (ELISA) ofregular insulin and insulin released from dissociated coacervates.

FIG. 12. Circular dichroism spectra of native insulin at different pH(n=3).

FIG. 13. Microscopy images of DgHBP-2 derived peptide coacervates (1mg/mL) loaded with BSA-FITC (0.1 mg/mL). Fluorescence image (left);light microscopy image (middle); merged microscopy image (right).

FIG. 14. Microscopy images of DgHBP-2 derived peptide coacervates (1mg/mL) loaded with GFP (0.1 mg/mL). Fluorescence image (left); lightmicroscopy image (middle); merged microscopy image (right).

FIG. 15. Microscopy images of DgHBP-2 derived peptide coacervates (1mg/mL) loaded with DOX (0.04 mg/mL) and MNP (0.002%). Top panel: withoutmagnetic field and bottom panel: with magnetic field. Aligned featureson the images indicate that MNPs are encapsulated within the droplets.Fluorescence image (left); light microscopy image (middle); mergedmicroscopy image (right).

FIG. 16. Recombinant DgHBP-1—amino acid sequence (SEQ ID NO:1 withadditional N-terminal M) and composition. DgHBP-1 possesses sequencefeatures characteristic for IDP exhibiting LLPS properties. It has lowsequence complexity that lacks Cys, Ile, Lys, and Met (added as astarting amino acid in recombinant form of the protein), Arg, and Trpresidues. The N-terminus contains negatively-charged residues (Glu, Asp)distributed along His-rich and Ala-rich clusters. The C-terminus lacksacidic residues and contains region of hydrophobic modular penta-repeats(marked in shades of blue and red) that are enriched mainly with Ala,Gly, His, Leu, Phe and Tyr residues.

FIGS. 17A-17C. ¹H-¹⁵N-HSQC spectra of DgHBP-1 at different pH values.(A) DgHBP-1 in the initial solution state at pH 3.3. (B) Dilute phaseafter LLPS at pH 6.5 (after sedimentation of coacervate micro-droplets).(C) Overlay of the two spectra. Spectra acquired at 298° K and a proteinconcentration of 2 mg/ml (130 μM).

FIG. 18. Residue-specific secondary structure propensity of DgHBP-1.Secondary shifts (ΔδC_(α)-ΔδC_(β)) indicate lack of secondary structureformation.

FIG. 19. Phase diagrams of DgHBP-1 at different pH values as a functionof protein concentration and ionic strength. The estimated boundarylines determine the pH at which LLPS (micro-droplets) was firstobserved. Ionic strength of the buffers (FIG. 35) adjusted with NaCl.

FIGS. 20A-20E. ¹H-¹⁵N Heteronuclear Single Quantum Coherence (HSQC)spectra of HBP-1 at different pH values ((A) pH 3.3, (B) pH 4.5, (C) pH5.5, (D) pH 6.5, (E) dilute phase after LLPS (pH 6.5)). Spectra acquiredat T=298° K and a protein concentration of 2 mg/ml (130 μM).

FIGS. 21A-21D. HSQC spectra of DgHBP-1 at different pH values ((A) pH3.3, (B) pH 4.5, (C) pH 5.5, (D) dilute phase after LLPS (pH 6.5))acquired at a lower protein concentration compared to initial conditions(FIGS. 20A-20E). Spectra acquired at T=298° K and a proteinconcentration of 0.5 mg/mL (32 μM).

FIGS. 22A-22D. HSQC spectra of DgHBP-1 at different pH values ((A) pH3.3, (B) pH 4.5, (C) pH 5.5, (D) dilute phase after LLPS (pH 6.5))acquired at a lower temperature (T=279° K) and a lower proteinconcentration of 0.5 mg/mL, 32 μM) compared to initial conditions (FIGS.20A-20E).

FIGS. 23A-23F. Analysis of LLPS properties of DgHBP-1 N- and C-terminalvariants and peptides. (A) Amino acid sequence representation of DgHBP-1protein fragment (SEQ ID NO:29). The repetitive region (G67-G145,corresponding to G66-G144 in SEQ ID NO:1) is presented with modularrepeats indicated with different black and white shades. Non-repetitiveN- and C-terminal regions are marked in white. (B) C-terminal variants(V1-C(SEQ ID NO:30) containing the whole repetitive region, and V2-Ctruncated at position G98 (SEQ ID NO:31)). (C) N- and C-variantsobtained by trypsin cleavage. (D) Synthetic peptides. The same colormarking was used for all peptides shown. Full amino acid sequences ofall proteins and peptides are presented in FIGS. 16 and 24A-C. Region ofthe DgHBP-1 sequence indicted in brackets. Variants that undergo LLPSmarked with “*”. (E) Phase diagrams (C: protein or peptide concentrationon x-axis and pH on y-axis) at low (0.1 M) and high (1 M) saltconcentrations, illustrating the conditions required to induce LLPS. Asindicated in the upper-left panel (DgHBP-1), at low proteinconcentration only one phase is present (soluble protein). When LLPSoccurs two phases co-exist, i.e protein rich phase (coacervatemicro-droplets/hydrogel) and protein depleted diluted phase (theboundary between two phases are drawn as a guide for the eye). Blackempty dots indicate pH and protein concentration at which opticalmicrographs presented in panel (F) were obtained. (F) Examples ofoptical micrographs taken after LLPS of all the variants and peptidesdescribed above and of DgHBP-1 (used as a control). Micrographs of V5-N,V6-N and V7-N represent hydrogels.

FIGS. 24A-24C. Sequences of DgHBP-1 protein variants. (A) V1-C and V-2C(were expressed with His-tag and TEV protease recognition site; SEQ IDNO:30 and 31). It was attempted to carry out proteolytic cleavage of theHis-tag; however we could not achieve efficient cleavage since theoptimum pH for the TEV protease was within the coacervation range of thevariants. It was observed that the variants containing the 6×His-TEV tagunderwent LLPS in the same way as full-length protein. Thus, weconcluded that the tag did not have any effect on their LLPS properties.(B) Variants obtained from trypsin cleavage of the protein mutantscontaining extra Lys residue. The mutants were created by geneticallyintroducing a single Lys residue into the protein sequence that servedas a recognition site for trypsin cleavage. Since the wild type HBP-1sequence completely lacks Arg and Lys, the addition of an extra Lysresidue allowed introducing a single specific cleavage site that couldbe precisely recognized by trypsin cleaving the protein into twofragments (referred here as N- and C-terminus fragments, e.g. Vx-N andVx-C, respectively). V3-N(SEQ ID NO:32), V4-N(SEQ ID NO:33), V5-N(SEQ IDNO:34), V6-N(SEQ ID NO:35), V7-N(SEQ ID NO:36), V3-C(SEQ ID NO:37), V4-C(SEQ ID NO:38), V5-C(SEQ ID NO:39), V6-C(SEQ ID NO:40), V7-C(SEQ IDNO:41) (C) Synthetic peptides with modular repeats GY-23 (SEQ ID NO:20);GA-25 (SEQ ID NO:42), GH-25 (SEQ ID NO:43).

FIGS. 25A-25C Recombinant DgHBP-2. (A) Amino acid sequence of theprotein with indicated trypsin sites (R in bold font) and GHGxY motif(dark grey) (SEQ ID NO:2) and additional N-terminal M, (B) DgHBP-2-N-and -C-terminal fragments (SEQ ID NO:44 and 45) obtained after trypsincleavage. (C) Optical micrographs after LLPS. Protein concentration 2mg/mL.

FIGS. 26A-26D LLPS properties of DgHBP-1 and -2 derived peptides. (A)Sequences and their ability to undergo LLPS. GY-25-V1 (SEQ ID NO:18),GY-25-V2 (SEQ ID NO:17), GY-20 (SEQ ID NO:19), GY-15-V1 (SEQ ID NO:48),GY-15-V2 (SEQ ID NO:49), GY-10-V1 (SEQ ID NO:47), GY-10-V2 (SEQ IDNO:50), GY-10-V3 (SEQ ID NO:51), GA-5-V1 (SEQ ID NO:27), GA-5-V2 (SEQ IDNO:46). (B) Phase diagrams of the peptides that exhibited LLPSproperties. (C) Sample morphology after LLPS by optical microscopy (leftmicrograph: gel; middle and right micrographs: micro-droplets). (D)Site-directed mutants of GY-23 peptide (SEQ ID NO:20, 52, 53) and theirLLPS ability. Color marking of DgHBP-1 modular repeats is identical tothe color-coding described in FIGS. 23A-F. All samples were tested inthe same conditions in various pH values and salt concentrations.

FIGS. 27A-27F NMR spectra of GY-23 peptide at different pH values(cross-peak trajectories marked with dashed lines). (A)¹H-¹⁵N-HMQCspectrum at initial conditions of pH 3.3. (B) Overlay of ¹H-¹⁵N-HMQCspectra acquired between pH 3.3 and 7 (pH 7: initiation of LLPS). (C,D)Overlay of ¹H-¹³C-HSQC spectra of aliphatic (C) and aromatic (D) sidechains at pH 3.3 and 7. The inset shows Tyr ¹H_(δ)-¹³C_(ζ) cross-peaksat pH 7. (E) Overlay of long-range ¹H-¹⁵N-HMQC spectra of His sidechains. The resonance assignments in the protonated state (pH 3.3) areindicated. (F) Long-range ¹H-¹⁵N-HMQC spectrum at pH 7 acquired within 5min after pH adjustment showing transient stabilization of Hisε-tautomer with characteristic resonance at ca. 250 ppm marked with thearrow. In the spectrum acquired after 30 min of pH adjustment, thiscross-peak was significantly attenuated (FIGS. 31A-31B). Spectraacquired at 298° K and a peptide concentration of 1.5 mM.

FIG. 28. Residue-specific secondary structure propensity of GY-23peptide. Secondary shifts (ΔδC_(α)-ΔδC_(β)) indicate lack of secondarystructure formation.

FIG. 29. ¹H-¹⁵N-HMQC spectra of GY-23 at different pH values. Bar graphsrepresent relative peak intensity for the assigned residues. Spectraacquired at 298° K and a peptide concentration of 1.5 mM.

FIGS. 30A-30C. SAXS and DLS of GY-23 peptide. (A) SAXS experimentalcurves of the peptide before and after coacervation. After LLPS, thedilute and coacervate-rich phases were measured followingcentrifugation. The q-3 power-law region of the scattering data ishighlighted, with the black line a guide for the eye. The calculated fitfor the peptide assemblies from the IFT method is also presented as acontinuous red line. (B) Corresponding p(r) profile calculated from theSAXS data in (A) using Eq. 1 (materials and methods). (C) Hydrodynamicdiameter (DH) measured by DLS of GY-23 in acetic acid (pH 3.3) and aftercoacervation (pH 7.0). Correlation functions showing the ‘raw’ data arepresented in FIGS. 34A-B.

FIGS. 31A-31B. Time-dependent comparison of the long-range ¹H-¹⁵N-HMQCspectra of His imidazole ring. (A) Spectrum acquired within 5 min afterpH adjustment to 7.0 (point of coacervation). (B) Spectrum acquiredwithin 30 min after pH adjustment. The characteristic resonance forε-tautomer at approximately 250 ppm in the N-dimension (marked with thearrow in panel (A) is present in the time frame of a few minutes shortlyafter pH adjustment to the coacervation point, indicating the transientnature of the interaction. Spectra acquired at T=298° K and a peptideconcentration of 1.5 mM.

FIGS. 32A-32B. Additional SAXS experiments with a synchrotron x-raysource. (A) SAXS experimental curve. The calculated fit for the peptideassemblies from the IFT method is presented as the full red line. (B)Corresponding p(r) profile calculated from the SAXS data in (A) usingEq. 1 (materials and methods). The arrow and the zoom-in view (inset)indicates that coacervate micro-droplets contain self-assembledsub-units of ca. 2 nm, which may be attributed to peptide oligomericunits.

FIGS. 33A-33D. Characterization of molecular interactions driving LLPSof GY-23 peptide by ssNMR. (A) Directly observed carbon spectrum of¹³C-selectively Tyr 5 and Tyr 23 labeled GY-23. (B)¹H-¹³Ccross-polarization (CP)-based 1D carbon spectrum of ¹³C selectively Tyr5 and Tyr 23 labeled GY-23. (C) DARR (100 ms mixing time). (D) HETCOR(100 μs mixing time) spectra of GY-23 (Tyr 5 and Tyr 23 labelled with¹³C and ¹⁵N). Correlations indicating Tyr-Tyr interactions are markedwith arrows.

FIGS. 34A-34B Correlation functions of the DLS measurements of GY-23peptide before and after coacervation (FIG. 33C) showing the intensitycorrelation function, C(τ) vs. the correlation time, τ. (A) Samplebefore coacervation (10 mM in acetic acid, pH 3.3). The curve with asingle decay at short correlation times represents a monomodaldistribution of rather small particles in the nanometer range. (B)Sample after LLPS (coacervation buffer, pH 7.0). The correlationfunction shows two decays at higher correlation times compared to (A)indicating the presence of two distinct populations of larger particles(coacervates).

FIG. 35. List of buffers used in LLPS studies of HBP-1 protein, itsvariants, and HBP peptides at different pH and ionic strength. Eachbuffer was prepared at 0.1 M, 0.5 M, and 1 M ionic strength (adjustedwith NaCl). Ionic strength defined as the sum of molar concentrations ofa salt component of a buffer and NaCl.

FIGS. 36A-36B. (A) Illustration of the coacervation and Doxorubicin(Dox)+magnetic nanoparticles (MNP) encapsulation process. Dox and MNPare first added to buffer, followed by additional of peptide stocksolution to initiate coacervation and encapsulation. (B) Dox+MNP loadedcoacervates can be directed away from health tissue and accumulated attumour site using directional magnetic field. Once the coacervatesreaches the target site, heat generated from MNP under alternatingmagnetic field (magnetic hyperthermia) induces the release of Dox. Heatgenerated can also enhance the cytotoxic activity of Dox.

FIG. 37. Dissociation of DgHBP-2 peptide coacervates (1 mg/mL) atdifferent temperature. This is to simulate the dissociation ofcoacervates by magnetic hyperthermia. Relative turbidity of coacervatesdecreased with increasing temperature. A rapid decrease was seen attemperature higher than 40° C. (magnetic hyperthermia for chemotherapyis usually between 42° C. to 48° C.).

FIG. 38. Microscopy images of DgHBP-2 peptide coacervates loaded withDox and MNP. Top panel: without magnetic field; Bottom panel: withmagnetic field. DgHBP-2 peptide coacervates can be loaded with bothDox+MNP and are magnetic-responsive.

FIG. 39. Encapsulation efficiency of different concentration ofDoxorubicin by DgHBP-2-peptide coacervates.

FIG. 40. Covalent crosslinking of catechol.

FIGS. 41A-41D. 4-Methylcatechol (4-MC)/NaIO₄ crosslinked coacervates.Micrographs of Dox+MNP loaded, 4-MC/NaIO₄ crosslinked coacervates. (Aand C) Light micrograph; (B and D) Fluorescence micrograph. (A and B)without magnetic field whereas (C and D) are with magnetic field. Theco-localisation of red fluorescence and 4-MC/NaIO₄ coacervates indicatesthat Dox is still retained within the coacervates after crosslinking.Under a directional magnetic field, the 4-MC/NaIO₄ coacervates alignedthemselves along the magnetic field and formed strings of coacervates.

FIGS. 42A-42B Release of Dox from 4-methylcatechol/NaIO₄ crosslinkedcoacervates at different temperature ((A) 20 min, (B) 16 hours). Therate of Dox release increases with increasing temperature. At 20 mincrosslinking, there is −40% leakage of Dox from coacervates over 48hours, whereas after 16 hours of crosslinking, the leakage decreases to−25%.

FIGS. 43A-43B MTT assay is a colorimetric assay for assessing cellmetabolic activity, using metabolic rate to infer cell survivability.(A) 4-Methylcatechol/NaIO₄ crosslinked coacervates (B), Dox-loaded4-methylcatechol/NaIO₄ crosslinked coacervates(MTT:(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide),NIH3T3 fibroblast cells).

FIGS. 44A-44B. Relative cell viability of Dox-loaded 4-MC/NaIO₄coacervates treated HEPG2 cells. After heating, Dox is released from thecoacervates and reduces the viability of HEPG2 cells. (A) Dox-loadedcoacervates in PBS without heat treatment. (B) Dox-loaded coacervates inPBS were heated at 45° C. for 30 min, before adding to cells(measurements at 570 nm).

FIGS. 45A-45C. GFP-loaded coacervates (C) were used to determine ifcoacervates can enter HEPG2 liver cancer cells. Within 3 hours ofincubation (A), most of the GFP-loaded coacervates co-localized with theHEPG2 cells. After 24 hours of incubation (B), GFP-loaded coacervateswere still visible. The coacervates seem to have merged and becamebigger compared to 3 hour incubation.

FIG. 46. Endosomal staining of GFP-loaded coacervates treated HEPG2cells. Left column: z-stack merged image and right column: orthographicview. Green and red fluorescence does not co-localize, indicating thatcoacervates were not taken up through endocytosis process (Lysotracker:GFP (green) (coacervates), Hoechst 33342 (blue) (nucleus), LysoTrackerRed DND-99 (red) (endosomes/lysosomes), wheat germ agglutinin(yellow-orange) (cell membrane)).

FIG. 47. 3D image GFP-loaded coacervates treated HEPG2 cells. TheGFP-loaded coacervates were located between the cell membrane andnucleus, indicating that the coacervates had successfully entered thecell (Lysotracker: GFP (green) (coacervates), Hoechst 33342 (blue)(nucleus), LysoTracker Red DND-99 (red) (endosomes/lysosomes), wheatgerm agglutinin (yellow-orange) (cell membrane)).

FIG. 48. Heating of Dox+MNP loaded coacervates by alternating magneticfield (AMF). Under AMF, MNP and Dox+MNP loaded coacervates were heatedto more than 50° C., whereas PBS (without any MNP) was heated to only38° C.

FIGS. 49A-49B. Release of Dox from Dox+MNP loaded, 4-MC/NaIO₄coacervates under alternating magnetic field (AMF). (A) Release of Doxunder AMF over 40 min. (B) Pulse release of Dox over 5 times of AMFtreatment, with each treatment 20 mins long to reach a temperature of45° C.

FIGS. 50A-50E. Biodistribution of cy5.5 loaded DgHBP-2 peptidecoacervates in BL6 mouse. 100 μL of DgHBP-2 peptide coacervates wereinjected into tail vein. (A) Total radiant efficiency of the liver afterinjection. (B) Total radiant efficiency of other organs/tissue excludingliver. (C) Micrograph of cyc5.5 loaded coacervates. colocalization ofcy5.5 within the coacervates droplets indicate that cy5.5 has beenencapsulated successfully. Within 1 hour of injection, most of thecoacervates were cleared by liver, kidneys and spleen. Of the threeorgans, liver has the highest radiant, indicating that this is the mainroute of excretion (A, B, C). (D) To improve the half-lifebiodistribution, cy5.5 loaded coacervates were coated with bovine serumalbumin before injecting into BL6 mouse. There were some differencesbetween the biodistribution of the coated and non-coated coacervates,notably the kidney, heart and aorta. (E) Merged fluorescence and lightimages of dissected organs from coated and non-coated coacervatesinjected mouse.

FIG. 51. Proposed model of pH-dependent LLPS of HBP-derived peptides. AtpH 3-4 His residues are protonated, and the peptides form solubleoligomeric units due to electrostatic repulsion between positivelycharged His side chains. At pH 4-6 gradual deprotonation of His residuesoccurs, repulsive forces are weaker but still strong enough to keep thepeptide oligomers soluble. At pH 6-7 transient interactions take placebetween His and Tyr residues located within GHGxY repeats leading tospecific peptide-peptide interactions that act as nuclei for LLPS.Further increase of pH above 7 leads to Tyr-Tyr inter-molecular stackingand intra-molecular interaction of hydrophobic residues that alltogether trigger LLPS and the formation of micro-droplets. If thecentral domain of the peptide is enriched with the hydrophobic motifGAGFA or with the His-rich motif GHGLH, LLPS is driven by the samesequence of molecular events but eventually leads to the formation ofeither a hydrogel or coacervate micro-droplets, respectively.

DETAILED DESCRIPTION

Compositions for delivery of an active agent, such as a pharmaceuticalor diagnostic agent, may include a peptide coacervate, said peptidecoacervate comprising peptides derived from histidine-rich proteins, andsaid active agent, wherein the active agent is encapsulated in thecoacervate as well as methods of manufacture thereof and methods of usethereof.

“Coacervate”, as used herein, has the meaning as commonly understood inthe art and discussed in the background section. Accordingly,coacervates are two-phase liquid compositions, i.e. exhibiting aliquid-liquid phase separation (LLPS), comprising or consisting of aconcentrated macromolecule-rich (or coacervate) phase and a dilutemacromolecule-depleted phase. The two phases of the peptide coacervatesare one peptide-rich coacervate phase and one dilute peptide-depletedphase. The peptide-rich coacervate phase is also referred to herein as“peptide colloid” or “peptide coacervate droplets”.

“Histidine-rich proteins”, as used herein, relates to proteins thatinclude at least one histidine residue and overall have a comparablyhigh amount of residues of the amino acid histidine (His or H). This maymean that the histidine content of a given protein is above 3%, forexample greater than 5% or greater than 10% or greater than 12%,relative to the total number of amino acids in the peptide sequence.

“Protein”, as used herein, relates to polypeptides, i.e. polymers ofamino acids connected by peptide bonds, including proteins that comprisemultiple polypeptide chains. A polypeptide typically comprises more than50, for example 100 amino acids or more.

“Peptide”, as used herein, relates to polymers of amino acids. Invarious non-limiting embodiments, the peptides may include only aminoacids selected from the 20 proteinogenic amino acids encoded by thegenetic code, namely glycine, alanine, valine, leucine, isoleucine,phenylalanine, proline, serine, threonine, asparagine, glutamine,tyrosine, tryptophan, histidine, arginine, lysine, aspartic acid,glutamic acid, cysteine, and methionine. These amino acids are alsodesignated herein by their three or one letter code. The peptides may bedipeptides, tripeptides or oligopeptides of at least 4 amino acids inlength. Typical lengths for the peptides may range from at least about 5amino acids to about 50 amino acids in length, for example at least 10,12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids inlength, the upper limit for example being 40, 35 or 30 amino acids.

“Encapsulate”, as used herein in relation to the active agent, meansthat the active agent is entrapped in the peptide coacervate phase, forexample the coacervate droplets formed by the peptides. Said entrapmentmay be such that the active agent is completely surrounded by peptidesforming the coacervate phase but also includes embodiments, where theactive agent is at least partially exposed on the surface of therespective coacervate phase, for example by being tethered to thecolloidal phase via a certain group or moiety.

“About”, as used herein in connection with a numerical value means saidvalue ±10%, for example ±5%.

In various embodiments, the composition is an aqueous liquid two phaseformulation. “Aqueous”, as used in this context, means that the dilutephase is mainly water, i.e. comprises at least 50 vol. % water. Invarious embodiments, the composition may use water as the only solvent,i.e. no additional organic solvents, such as alcohols, are present. Inother embodiments, the composition is an aqueous composition thatadditionally contains one or more solvents other than water, with waterhowever being the major constituent, i.e. being present in an amount ofat least 50, at least 60, at least 70, at least 80, at least 90, atleast 95 or 99 vol. %.

In such embodiments, the coacervate colloidal phase comprises thepeptides derived from histidine-rich proteins in form of colloidsencapsulating the active agent. In some embodiments, the colloidal phasehas the form of droplets having a substantially spherical shape with adiameter ranging from about 0.2 to about 5 μm. The diameter of thesubstantially spherical shape may be the “equivalent spherical diameter(ESD)” referring to the diameter of a perfect sphere of equivalentvolume as the potentially irregularly shaped droplet. For example, thedroplet may have an ellipsoid shape, and the equivalent sphericaldiameter would then be the diameter of a perfect sphere of exactly thesame volume. Each of the droplets is made up of the coacervate-formingpeptides and, in various embodiments, is homogeneous in that it has nodistinct core-shell morphology, but rather is a colloidal particle withno peptide gradient over its radius. In alternative or additionalembodiments, the coacervate phase may take the form of a condensedhydrogel.

The dilute aqueous phase may include a water-based liquid, as describedabove. It is peptide-depleted in that the majority of the peptides arelocated in the colloidal phase, e.g. 80 wt.-% or more of the peptidesare present in the colloidal phase, for example at least 85, at least90, at least 95, at least 96, at least 97, at least 98, or at least 99wt.-%. The dilute phase may thus contain residual amounts of peptides inthe coacervate composition. As the coacervate formation is anequilibrium reaction, the exchange of peptides from the colloidal phaseto the dilute phase and vice versa may be dynamic. However, in variousembodiments, the above distribution applies.

In various embodiments, the histidine-rich proteins are histidine-richbeak proteins (DgHBPs), for example derived from the beak of a squid, inparticular the Humboldt squid (Dosidicus gigas). The histidine-richproteins may, for example, include or be, but not limited to, DgHBP-1(SEQ ID NO:1) and DgHBP-2 (SEQ ID NO:2), with the peptides derivedtherefrom being, in various embodiments, fragments thereof that compriseat least one copy of the peptide motif GHGXY, wherein X is valine (V),leucine (L) or proline (P).

As used herein, “peptides derived from histidine-rich proteins”generally refers to peptides that represent fragments or variants orboth of histidine-rich proteins, in particular histidine-rich proteinsthat naturally occur, for example in the Humboldt squid. The peptidesmay be produced by genetic engineering techniques as known to thoseskilled in the art. The peptides thus artificially produced mayrepresent amino acid stretches of the proteins they are derived from butdo not encompass the full native protein sequence. In variousembodiments, the derived peptides are N- and/or C-terminally truncatedfragments of the respective histidine-rich protein. In case of DgHBP-1and DgHBP-2, the peptides may include N-terminally truncated fragments,e.g. C-terminal fragments missing at least the N-terminus. In variousembodiments, these may also be C-terminally truncated such that theymiss one or more amino acids from the C-terminus. In various otherembodiments, the peptides are further modified in that they also lack,in addition to an N- and/or C-terminal truncation, amino acids withinthe sequence relative to the respective stretch in the templatesequence. Alternatively or additionally, the peptides may also compriseamino acid substitutions, deletions or insertions relative to theprotein sequence they are derived from.

The peptides may also be derived from histidine-rich proteins in thatthey comprise a sequence motif also occurring in said proteins with therest of their sequence optionally being different from that of thehistidine-rich protein they are derived from.

When reference is made to sequence differences or sequence identity,this means that in a given peptide molecule the respective amino acid ata given position is identical to the amino acid in a referencepeptide/protein at the corresponding position. The level of sequenceidentity is given in % and can be determined by an alignment of thequery sequence with the template sequence.

The determination of the identity of amino acid sequences is achieved bya sequence comparison. This comparison or alignment can, for example, bebased on the BLAST algorithm well-established and known in the art (see,e.g., Altschul et al. (1990) J. Mol. Biol. 215, 403-410; and Altschul etal. (1997) Nucleic Acids Res., 25, 3389-3402) and is in principlecarried out by aligning stretches of amino acids in the peptidesequences with each other.

Such a comparison allows determining the identity of two sequences andis typically expressed in % identity, i.e. the portion of identicalamino acid residues in the same or corresponding positions. If notexplicitly stated otherwise, the sequence identities defined hereinrelate to the percentage over the entire length of the respectivesequence, i.e. typically the reference sequence. If the referencesequence is 20 amino acids in length, a sequence identity of 90% meansthat 18 amino acids in a query sequence are identical while 2 maydiffer. In a non-limiting embodiment, the peptides include at least thesequence having GHGXY within the peptide sequence; all other amino acidswithin the sequence may be changed without affecting the functionalityof the peptide. X of the GHGXY sequence may be valine (V), leucine (L),or proline(P) in another non-limiting embodiment.

As the peptides include fragments and variants of histidine-richproteins that do not occur in nature and have typically beenartificially produced, the peptides are, in various embodiments,artificial peptides, such as those created by genetic engineeringtechniques, recombinant peptides, and the like known to those skilled inthe art.

In various embodiments, the peptide has a minimum length of 16 aminoacids, for example 18 or 20 amino acids, and comprises at least twosequence motifs GHGX¹Y, for example GHGLY, or at least one sequencemotif GHGX¹Y, for example GHGLY (SEQ ID NO:46), and at least onesequence motif GHGLX⁴, for example GHGLH (SEQ ID NO:28) or GHGLG (SEQ IDNO:54).

In non-limiting embodiments, the peptides may include at least twocopies of the motif GHGX¹Y, for example GHGLY, separated by a spacercomposed of the motifs (i) GX²GX³A, for example GAGFA (SEQ ID NO:27),(ii) GFA and/or (iii) GHGLX⁴, for example GHGLH or GHGLG, the spacerhaving a minimum length of 13 amino acids, for example GX²GX³AGHGLX⁴GFA.Alternatively, the peptides of may include at least four copies of themotif GHGX¹Y, for example GHGLY.

In various embodiments, the peptides derived from histidine-richproteins comprise the amino acid sequence

(GHGX¹Y)_(a)[(GX²GX³A)_(b)(GHGLX⁴)_(c)(GFA)_(d)]_(f)(GHGX¹Y)_(a)

wherein X¹ is valine (V), leucine (L) or proline (P), X² is alanine (A)or proline (P); X³ is phenylalanine (F) or tyrosine (Y), X⁴ is leucine(L), histidine (H), tyrosine (Y) or glycine (G),

each a is 0 or an integer≥1;

each b is 0 or an integer≥1;

each c is 0 or an integer≥1;

each d is 0 or an integer≥1;

f is an integer≥1;

with the sum of all a being ≥2 and the sum of all a+b+c+d is ≥4.

In the above sequence and all further sequences disclosed below, aminoacids are identified by their one letter code. Thus, G stands forglycine, H stands for histidine, L stands for leucine, Y stands fortyrosine, etc. The peptides are also shown in the conventional way, i.e.in the N- to C-terminal orientation. The individual amino acids arecovalently coupled to each other by peptide bonds.

In the above peptides, the minimum sequence motif that is present is twomotifs GHGX¹Y, for example GHGLY, with the peptide being at least 16amino acids in length (sum of all a=2 and sum of all d=2).

The upper limit in peptide length may be 50 amino acids, for example upto 40, up to 35 or up to 30 amino acids.

Exemplary peptides encompassed by the above sequence are describedbelow.

In addition to the above sequence motifs, the peptides may compriseadditional amino acids on their N- or C-terminal end or on both, forexample 1-10 or 1-5 additional amino acids. In various non-limitingembodiments, the peptides may additionally comprise a C-terminaltryptophan residue (W). In various non-limiting embodiments, thepeptides may include only 1-5 additional amino acids on their termini inaddition to the above sequence motifs. Alternatively, the peptides mayinclude only one or more of the above sequence motifs in anothernon-limiting embodiment.

All peptides disclosed herein may be additionally modified by non-aminoacid moieties, such as lipid or carbohydrate or other organic orinorganic moieties, including PEGylation, farnesylation, and the like.These modifications may impart additional desirable properties, forexample increased hydrophobicity and the like.

In specific embodiments, the peptides derived from histidine-richproteins may include an amino acid sequence, such as but not limited to:

-   -   (1) (GHGXY)_(n),    -   wherein X is valine (V), leucine (L) or proline (P), and n is        ≥4;    -   (2) [(GHGXY)_(n)(GAGFA)_(m)]_(i)GHGXY    -   wherein X is valine (V), leucine (L) or proline (P); n is ≥1; m        is ≥1; and i≥2;    -   (3) [(GHGXY)_(n)(GHGLH)_(m)]_(i)GHGXY    -   wherein X is valine (V), leucine (L) or proline (P); n is ≥1; m        is ≥1; and i≥2;    -   (4) (GHGXY)_(n)(GAGFA)_(m)GHGXY    -   wherein X is valine (V), leucine (L) or proline (P); n is ≥1;        and m is ≥2; and    -   (5) (GHGXY)_(n)(GHGLH)_(m)GHGXY    -   wherein X is valine (V), leucine (L) or proline (P); n is ≥1;        and m is ≥2;

Non-limiting peptides may include an amino acid sequence, such as butnot limited to,

(SEQ ID NO: 3) (i) GHGXY GHGXY GHGXY GHGXY GHGXY W (SEQ ID NO: 4)(ii) GHGXY GHGXY GHGXY GHGXY GHGXY  (SEQ ID NO: 5)(iii) GHGXY GAGFA GHGXY GAGFA GHGXY  (SEQ ID NO: 6)(iv) GHGXY GHGLH GHGLH GHGLH GHGXY  (SEQ ID NO: 7)(v) GHGXY GAGFA GAGFA GAGFA GHGXY  (SEQ ID NO: 8)(vi) GHGXY GHGXY GHGXY GHGXY  (SEQ ID NO: 9)(vii) GHGXY GAGFA GHGLH GFA GHGXY  (SEQ ID NO: 10)(viii) GHGXY GAGFA GHGLH GAGFA GHGXY  (SEQ ID NO: 11)(ix) GHGXY GHGLH GAGFA GHGLH GHGXY  (SEQ ID NO: 12)(x) GHGXY GAGFA GAGFA GHGLH GHGXY  (SEQ ID NO: 13)(xi) GHGXY GHGLH GAGFA GAGFA GHGXY  (SEQ ID NO: 14)(xii) GHGVY GHGVY GHGPY GHGPY GHGLY W (SEQ ID NO: 15)(xiii) GHGVY GHGVY GHGPY GHGPY GHGLY  (SEQ ID NO: 16)(xiv) GHGLY GAGFA GHGLY GAGFA GHGLY  (SEQ ID NO: 17)(xv) GHGLY GHGLH GHGLH GHGLH GHGLY  (SEQ ID NO: 18)(xvi) GHGLY GAGFA GAGFA GAGFA GHGLY  (SEQ ID NO: 19)(xvii) GHGLY GHGLY GHGLY GHGLY  (SEQ ID NO: 20)(xviii) GHGLY GAGFA GHGLH GFA GHGLY  (SEQ ID NO: 21)(xix) GHGLY GAGFA GHGLH GAGFA GHGLY  (SEQ ID NO: 22)(xx) GHGLY GHGLH GAGFA GHGLH GHGLY  (SEQ ID NO: 23)(xxi) GHGLY GAGFA GAGFA GHGLH GHGLY  (SEQ ID NO: 24)(xxii) GHGLY GHGLH GAGFA GAGFA GHGLY 

All the above peptides may comprise additional N- and/or C-terminalamino acids, in particular a C-terminal W, if not already present.Furthermore, all peptides have, in various embodiments, a maximum lengthof 50 amino acids, for example 40 amino acids and less.

In non-limiting embodiments, the peptides may include, consistessentially of, or consist of the amino acid sequences set forth above.

The peptides may be synthesized using any conventional method known forpeptide synthesis, including chemical synthesis and recombinantproduction. Suitable methods are well-known to those skilled in the artand may be selected using their routine knowledge.

It has been found that the above peptides form coacervates readily, inparticular under neutral conditions, i.e. pH values of 7 and higher.Stable solutions of these peptides without any distinct phase separationcan be formed at low pH, for example less than 4. In variousembodiments, the peptides may be prepared as stock solutions in slightlyacidic solutions, such as 1-100 mM, for example about 10 mM acetic acidor other suitable weak acids.

In various embodiments, the pH of the composition is 7.0 or higher, forexample ranging from about 7.4 to about 9.5. These pH values ensure thatthe colloidal phase remains stable.

In various embodiments, the peptide coacervate, i.e. the coacervatephase formed, can be covalently crosslinked. The crosslinking may beachieved by use of a suitable crosslinker. As the peptides disclosedherein typically comprise at least one tyrosine residue, the hydroxylgroups thereof may be used for crosslinking. Suitable crosslinkingagents include, without limitation, those that comprise catecholmoieties, for example and without limitation 4-methylcatechol (4-MC). Insuch cases, crosslinking may occur by a redox reaction between thecatechol moieties and aromatic hydroxyl groups, such as the tyrosinehydroxyl groups, heteroaryl groups, such as histidine imidazole groups,and amine groups, such as lysine, arginine, asparagine or glutamineamine groups. In one specific embodiment, the peptide coacervate iscrosslinked with 4-methylcatechol (4-MC) and sodium periodate (NaIO₄).

The active agent may, for example, be a pharmaceutical or diagnosticagent. Generally, it may be or include, but not limited to, proteins,(poly)peptides, carbohydrates, nucleic acids, lipids, chemical compoundsand nanoparticles. Suitable nanoparticles include those, such as but notlimited to, metal nanoparticles, metal oxide nanoparticles andcombinations thereof. The nanoparticles may be magnetic nanoparticles.“Nanoparticles”, as used herein, refer to particles that havedimensions, such as ESD, in the nanometer range, typically up to 500 nm,for example up to 250 or up to 100 nm. The nanoparticles may besubstantially spherical in shape in a non-limiting embodiment. “Chemicalcompounds”, as used in this context, relates in particular to smallmolecules, i.e. organic compounds with a molecular weight of 1000 g/molor less, such as 750 g/mol and less. This group of compounds includes,for example, known small molecule pharmaceutical compounds, such asdoxorubicin. A pharmaceutical agent from the group of (poly)peptidesincludes insulin and other peptide hormones.

In various embodiments, the pharmaceutical or diagnostic agent may be orinclude, but not limited to, RNA oligonucleotides or variants thereof,such as those used in CRISPR/Cas9 or other genome-editing systems, smallmolecules, antibodies or antibody-like molecules, and the like.

The pharmaceutical compound may comprise or be insulin, in particularhuman insulin, either alone or in combination with the enzyme glucoseoxidase. The enzyme glucose oxidase naturally occurs in honey and isalso produced by a variety of fungi. It catalyzes the oxidation ofglucose in the C1 position and yields gluconic acid. In aqueoussolutions, gluconic acid undergoes an internal esterification reactionto form gluconolactone.

In various other embodiments, the pharmaceutical agent comprises or isan anti-cancer agent, such as a cytostatic agent, for exampledoxorubicin. Doxorubicin may be encapsulated either alone or incombination with magnetic nanoparticles.

Generally, the active agent, such as insulin or doxorubicin, may beencapsulated alone or co-encapsulated together with a release agent thatfacilitates release of the active agent from the coacervate. Examples ofsuch release agents have been disclosed above and include magneticnanoparticles and glucose oxidase in a non-limiting embodiment.Depending on the type of release agent used, the release mechanism maydiffer. One type of release agents leads to an acidification of theenvironment of the colloidal phase, with the lowering of the pHtriggering the dissolution of the coacervate phase. The acidificationmay be dependent on the presence of an initiator or substrate, in thecase of glucose oxidase, glucose. The addition of glucose or theincrease in glucose concentration may thus lead to sufficientacidification to facilitate release of the encapsulated agent, forexample insulin. Other types of release agents cause heat development inthe vicinity of the colloidal phase that also effects release of theencapsulated agent. Such heat development may, for example, be achievedby magnetic nanoparticles and exposure to a magnetic field.

Further release mechanisms, such as peptide degradation by use of apeptidase, may be possible and can be selected by those skilled in theart dependent on the intended use.

Generally, the release may be a burst release where essentially thetotal load of the active agent is released over a short time span or maybe a sustained release where the release occurs over a prolonged periodof time. In general, the release may occur within several minutes up toseveral days or weeks. The release may also be step-wise in that uponexposure to certain conditions the release starts but stops when theconditions are no longer met. It can then start again once theconditions for release are again met. Such conditions that may be variedto facilitate a step-wise or need dependent release may include, but arenot limited to, glucose concentration and magnetic field exposure.

The composition may be a pharmaceutical or diagnostic formulation foradministration to a subject. Such formulations may additionally compriseall the known and accepted additional components for such applications.These include auxiliaries, carriers and excipients that arepharmaceutically or diagnostically acceptable, for example varioussolvents, preservatives, dyes, stabilizers and the like. Suchformulations may additionally comprise further active agents that arenot encapsulated in the coacervate phase. In various embodiments, suchcompositions are liquid compositions, including gels and pastes.“Liquid”, as used herein, particularly refers to compositions that areliquid under standard conditions (20° C. and 1013 mbar). In variousembodiments, such liquid compositions are pourable. The compositions maybe in single dose or multi dose form. Suitable forms and packagingoptions are well known to those skilled in the art.

The compositions can be adapted for administration to a mammaliansubject, for example a human being.

For the encapsulation, an aqueous solution of the coacervate-formingpeptides can be used. As described above, it is possible to dissolve thepeptides in an aqueous solution if the pH is low enough. Accordingly,the peptides can be dissolved in aqueous acetic acid, for example of aconcentration of 1 to 100 mM, such as 10 mM. Other acids may be equallysuitable, as long as they do not hydrolyze the peptide bonds or are usedin concentrations low enough to avoid hydrolysis of the peptides. Inthese embodiments, the pH of the aqueous solution of thecoacervate-forming peptides may be below 7, for example below 6.5 orbelow 6.0 or below 5.5 or below 5.0 or below 4.5 or below 4.0. The pH ishowever, in various embodiments, higher than 0, for example 1 or higher,such as 2 or higher.

For forming the coacervate and at the same time encapsulating the activeagent, the solution of the coacervate-forming peptides is combined withthe active agent and coacervate formation is induced. The induction ofcoacervate formation is typically induced by increasing the pH of theresulting solution containing both the coacervate-forming peptides andthe active agent, as well as optional additional components andauxiliaries. The pH may be increased to values of 6.0 or more, 6.5 ormore, 7.0 or more, but, in various embodiments, not higher than 10.0.The pH increase may be achieved by adding an alkaline agent to thesolution. In case the active agent is provided in form of an aqueoussolution, too, said solution may have a pH>7 and thus effect coacervateformation. To maintain the pH in a range high enough upon combination ofthe two solutions, the solution of the active agent may be buffered withsuitable buffering agents, such that the combined aqueous solutions ofthe active agent and the coacervate-forming peptides retain a pH>7.

The concentration of the coacervate-forming peptides in the aqueoussolution may range from 2 to 100 mg/mL. To allow efficient coacervateformation, in various embodiments, the concentration of thecoacervate-forming peptides in the aqueous solution after addition ofthe active agent is greater than 0.3 mg/mL.

After the coacervate has been formed, it may be an aqueous liquid twophase formulation, as described above, i.e. a composition comprising (1)a coacervate colloidal phase comprising the peptides derived fromhistidine-rich proteins and the active agent; and (2) a dilute aqueousphase.

The coacervates formed in the above-described processes may have theform of droplets, for example microdroplets, having a substantiallyspherical shape with a diameter ranging from about 0.2 to about 5 μm, ormay take the form of a condensed hydrogel.

In the methods for the delivery of an active agent, such as apharmaceutical or diagnostic agent, the provided compositions comprisinga peptide coacervate may be exposed to or subjected to conditions thatfacilitate the release of the active agent from the coacervate phase.Said release may be facilitated by dissolution of the peptides of thecoacervate phase, for example reversing the formation process bydecreasing the pH, or degradation or disruption of the coacervate phaseby suitable means. Some of the release mechanisms have been describedabove. Additional release mechanisms may include the use of surfactantsor denaturing agents that disrupt the formed phases.

In various embodiments, the conditions that trigger the release of thepharmaceutical or diagnostic agent may be or include, but not be limitedto, elevated temperatures, pH changes, exposure to release agents, suchas enzymatic agents that degrade peptides, denaturing agents orsurfactants, and combinations thereof.

Methods for treating or diagnosing a condition or disease or disorder ina subject in need thereof is also disclosed, wherein the compositionsdescribed above may be used in the treatment and/or diagnosis. Suchmethods of treatment also include methods where a disease, condition ordisordered is managed, for example in that the symptoms or effects arealleviated. The treatment methods thus also include methods for themanagement of diabetes, wherein the insulin deficiency in the patient isremedied by controlled release of insulin upon certain stimuli.

In such methods, the compositions described herein and comprising apeptide coacervate and a pharmaceutical or diagnostic agent, wherein thepharmaceutical or diagnostic agent is encapsulated in the coacervate areadministered to said subject. The administration may make use of anysuitable administration route including oral administration orparenteral administration, for example intravenous, intramuscular,subcutaneous, epidural, intracerebral, intracerebroventricular, nasal,intraarterial, atraarticular, intracardiac, intradermal, intralesional,intraocular, intraosseous, intravitreal, intraperitoneal, intrathecal,intravaginal, transdermal, transmucosal, sublingual, buccal, andperivascular.

The administration may be systemic or localized, e.g. topically.

After administration, the release of said pharmaceutical or diagnosticagent from the coacervate may be facilitated by exposing the coacervateto conditions that trigger the release of the pharmaceutical ordiagnostic agent. Said exposure may occur automatically due toconditions in the body of the patient, such as metabolic action, or maybe triggered externally by applying a stimulus to the patient that leadsto release of the encapsulated agents, such as exposure to a magneticfield.

The conditions that trigger the release of the pharmaceutical ordiagnostic agent may generally be selected from those disclosed abovefor the delivery methods. The subject may be a mammal, for example ahuman.

In non-limiting embodiments of these methods for the treatment of adisease or disorder, the subject is a human afflicted by diabetes,wherein the pharmaceutical or diagnostic agent is insulin, wherein thecoacervate further comprises encapsulated glucose oxidase, and whereinrelease of the glucose oxidase is facilitated by an increase in glucoseconcentration and the resulting acidification of the coacervate. In suchembodiments, an increase in glucose concentration that may occur in thepatient after consumption of food leads to increased levels of glucosepermeating into the coacervate phase. In the coacervate phase, theentrapped glucose oxidase enzyme catalyzes the oxidation of glucose togluconic acid which leads to acidification of the microenvironment ofthe enzyme. As a result of the lowered pH, the coacervate phase/dropletloses its structural integrity and the release of the also encapsulatedinsulin is facilitated.

In further non-limiting embodiments, the subject is a human afflicted bycancer, wherein the pharmaceutical agent is doxorubicin, wherein thecoacervate further comprises encapsulated magnetic nanoparticles, andwherein release is facilitated by exposure of the subject to a magneticfield resulting in a temperature increase in the coacervate. In suchembodiments, the subject or a body region of the subject may be exposedto magnetic fields that lead to a temperature increase in the vicinityof the magnetic particles and as a result loss of the structuralintegrity of the coacervate phase that then releases the doxorubicin.

In non-limiting embodiments, the cancer may be liver cancer, coloncancer, lung cancer, prostate cancer, breast cancer, and the like.

It is understood that release facilitated by glucose is not limited toinsulin release but may also be used for release of other agents.Similarly, release by exposure to magnetic fields may also be used fordifferent agents than doxorubicin, such as other anti-cancer agents,contrast agents used for imaging methods and the like.

Additional applications of the compositions and methods will beidentifiable by the person skilled in the art. The compositions andmethods herein disclosed are further illustrated in the followingexamples, which are provided by way of illustration and are not intendedto be limiting the scope of the present disclosure.

EXAMPLES Materials and Methods Turbidimetry for DgHBP-2 PeptideCoacervation Studies

10 mg of DgHBP-2 peptide (GL Biochem) were dissolved in 10 mM aceticacid to make 10 mg/mL of stock solution. During coacervation studies, 10μL of stock solution were added to 90 μL of buffer to make a finalpeptide concentration of 1 mg/mL. Coacervation studies at differentbuffer conditions (pH 3.5 to 10, 0.1 to 1.0M ionic strength) was thenmeasured at 600 nm by UV-vis spectrophotometry. Relative turbidity iscalculated as¹:

100−100*[10^(−A600)]

where A600 is the absorbance value at 600 nm wavelength.

Dynamic Light Scattering (DLS)

DLS measurements were performed on ZetaPALs (Brookhaven InstrumentsCorporation) equipped with a 35 mW red diode laser (640 nm wavelength).100 μL of DgHBP-2 peptide coacervates containing 1 mg of peptide wasprepared for the measurement. The scattering angle was set to be 90° andeach sample was measure 5 times.

Fluorescence and Optical Microscopy

In Example 1 below, microscope images were taken using an invertedmicroscope with 63× oil Immersion lens (Zeiss Axio observer z1). Imageswere processed with Zen microscope and imaging software (Zeiss, blueedition). For fluorescence imaging, 1 mg/mL of DgHBP-2 peptide was usedto encapsulate 0.1 mg/mL of RITC-GOx and FITC-insulin.

In Example 2 below, phase separation behavior of protein variants andpeptides was studied using a Zeiss Axio Scope A1 microscope (Carl ZeissPte Ltd., Germany) in the reflection mode, with differentialinterference contrast (DIC) filters. Images were taken with an AxioCamMRc 5 camera under the control of AxioVision software.

Labelling of Insulin and Glucose Oxidase (GOx)

Fluorescence isothiocyanate dyes (Fluorescein or Rhodamine B, (SigmaAldrich)) were dissolved in acetone to a concentration of 3 mg/mL. 200μL of dye solution was added slowly to 1 mg of insulin (Sigma Aldrich)dissolved in 0.1 M phosphate buffer pH 7 or GOx (Sigma Aldrich)dissolved in 0.1 M carbonate buffer pH 8.5. The reaction mixture wasincubated at room temperature with constant stirring for 16 hours.During the reaction, the tubes were covered with aluminium foil toprevent photobleaching. After incubation, the reaction mixture wassubjected to PD-10 gel filtration column (GE healthcare life sciences)for separating unreacted dyes from the labelled Insulin/GOx. Thelabelled insulin and GOx were then eluted with phosphate buffer.

Insulin and GOx Encapsulation

Insulin and GOx were added to buffer solution before coacervation totheir respective concentrations. DgHBP-2 peptide stock solution (10mg/mL) was subsequently added to the buffer solution at 1:9 ratio toinduce coacervation and encapsulation. The final mixture was then mixedby pipetting.

MTT Assay

NIH3T3 mouse fibroblasts were used to estimate the cytotoxicity ofDgHBP-2 peptide and coacervates. The fibroblasts were cultivated usingDulbecco's modified eagle medium supplemented with 10% of fetal bovineserum, 100 units/mL of penicillin and 100 μg/mL of streptomycin in a 37°C. incubator with 5% CO₂. 100 μL of cells with a cell density of 1×10⁴cells/mL were seeded onto a 96-well plate (Thermo Fisher Scientific).After 24 hours, the cell culture media was substituted with 100 μL offresh media containing different concentration of peptide orinsulin-loaded coacervates. After another 24 hours, 10 μL of MTTsolution (concentration: 5 mg/mL) was added into each well. Cell culturemedia and the excess MTT was then removed after 4 hours of incubation.100 μL of DMSO was subsequently added into each well to dissolve theformazan crystals. The dissolved crystals were then measured at 570 nmon a 96-well microplate reader (Infinity M200, Tecan). Relative cellviability is calculated as:

$\frac{\left( {A_{t} - A_{b}} \right)}{\left( {A_{c} - A_{b}} \right)}*100\%$

where A_(t), A_(c), and A_(b) represent absorbance values of wellscontaining tested cells, control cells and DMSO, respectively.

In Vitro Release Assay (Timepoint Assay)

50 μL of 1 mg/mL of DgHBP2-peptide coacervates were loaded with 0.1mg/mL of FITC-insulin and/or 0.01 mg/mL of GOx. The coacervates werediluted to a final volume of 150 μL using phosphate buffer (pH 7.5,0.1M), and placed within individual dialysis tubes (12-14 kDa cut-off,Millipore). The coacervates was then dialysed against 1 mL of phosphatebuffer with/without 4 mg/mL of glucose for 48 hours (37° C., 250 rpm).The fluorescence of the dialysed buffer was measured using afluorescence spectrophotometer (Fluorolog, HORIBA Jobin Yvon) andreplaced with new buffer at different time points.

In Vitro Release Assay (Alternating Glucose Concentration Assay)

The GOx+insulin coacervates were prepared similarly as above. Thecoacervates was then dialysed against 1 mL of phosphate buffer withglucose for 9 hours (37° C., 250 rpm). The concentration of glucose wasalternated between 1 and 4 mg/mL every 1.5 hours. The fluorescence ofthe dialysed buffer was then measured using a fluorescencespectrophotometer (Fluorolog, HORIBA Jobin Yvon) at every 1.5 hours.

Circular Dichroism Spectroscopy

DgHBP-2 peptide and insulin were prepared at 1 mg/mL and 0.1 mg/mLrespectively. Circular dichroism (CD) spectra was collected using aquartz cuvette of 0.2/0.5 mm optical pathlength. Data collection wasdone on AVIV 420 Circular Dichroism spectrometer with the followingparameters: average of three scans per experiment between wavelengthrange from 190 to 250 nm, 1.0 nm wavelength steps with 1.00 nm bandwidthand 0.1 s averaging time. Each experiment was repeated for 3 times andaveraged to obtain the final spectrum. The final spectrum was thensmoothed and plotted using originPro9.1.

Liquid-Liquid Phase Separation

Liquid-liquid phase separation properties of protein variants andpeptides at different buffer conditions were assessed using the methoddescribed in (Tan et al, Nat. Chem. Biol. 11, 488-495). Briefly,protein/peptide stock solution (10 mg/mL in 10 mM acetic acid, pH 3.3)was added to a buffer solution in a volume ratio 1:5 (protein/peptidestock:buffer). The mixture was then pipetted onto a microscopy glassslide and imaged using the optical microscope.

Solution-State NMR Spectroscopy

Sample preparation—lyophilized samples were dissolved in 10 mM aceticacid (pH 3.3) containing 10% D₂O and 0.2 mM DSS prior the NMRexperiments. 0.5 M NaOH was used for pH adjustment during pH titrationexperiments.

NMR experiments—three-dimensional BEST-TROSY HNCO, HNCA, HN(CO)CA,HNCACB, HN(CO)CACB, HN(CA)CO experiments (Solyom, Z. et al. (2013)BEST-TROSY experiments for time-efficient sequential resonanceassignment of large disordered proteins. J. Biomol. NMR 55, 311-321) forDgHBP-1 protein backbone assignment were recorded on a 700 MHz BrukerAdvance III NMR spectrometer equipped with 5 mm z-gradient TXI cryoprobeoperating at 298 K. The spectra were acquired using non-uniform sampling(NUS) with 30% amount of sparse sampling. Processing of the NUS spectrawas performed using MDDNMR program (Orekhov, V. Y. & Jaravine, V. A.(2011) Analysis of non-uniformly sampled spectra with multi-dimensionaldecomposition. Prog. Nucl. Magn. Reson. Spectrosc. 59, 271-292)implemented in TopSpin 3.5 (Bruker) software. Backbone assignment wascarried out using CARA software (http://cara.nmr.ch/). ¹H-¹⁵N-HMQCspectra at different pHs were acquired using SOFAST-HMQC pulse program(Schanda, P. & Brutscher, B. (2005) Very fast two-dimensional NMRspectroscopy for real-time investigation of dynamic events in proteinson the time scale of seconds. J. Am. Chem. Soc. 127, 8014-8015) on an800 MHz Bruker Advance III NMR instrument equipped with 5 mm QCI H/P/C/Nsolution cryoprobe, at 298 K.

Data for GY-23 backbone assignment were collected on the 800 MHzspectrometer. The same set of BESTTROSY experiments (as for DgHBP-1protein, expect of HN(CO)CACB) were recorded utilizing NUS with 10-30%amount of sparse sampling. Processing of the data and backboneassignment was performed as described above. Experiments during pHtitration: ¹H-¹⁵N-HMQC, ¹H-¹³C-HSQC, and long-range ¹H-¹⁵N-HMQC spectraof His side chains were acquired using standard pulse programs from theTopSpin 3.5 repository on the 700 MHz spectrometer. ¹⁵N- and¹³C-HSQC-NOESY with 500 ms mixing time were acquired on the 600 MHzBruker Advance III spectrometer equipped with 5 mm z-gradient TCIcryoprobe, at 298 K.

SAXS Example 1

Sample preparation—5.0 mg of lyophilized GY-23 peptide was dissolved in100 μL of 10 mM acetic acid (pH 3.3). Coacervation was induced by mixingof the peptide stock with the coacervation buffer (50 mM Tris-HCl, pH7.0 buffer, containing 1 M NaCl) in 1 to 5 volume ratio. Coacervate-richphase was collected by centrifugation (13000 g for 5 minutes at 25° C.)and transferred into a 1.5 mm quartz capillary together with somesupernatant to avoid drying. The position of the capillary was thenspecifically aligned to hit the coacervate-rich phase.

SAXS measurements—were performed on a Bruker Nanostar U (Bruker AXS,Karlsruhe, Germany) connected to a sealed-tube Cu anode X-ray sourceoperating at 50 kV and 600 μA (Incoatec IpSCu, Geest-hacht, Germany). AGöbel mirror was used to convert the divergent polychromatic X-ray beaminto a focused beam of monochromatic Cu Kα radiation (λ=0.154 nm). Thebeam size was 0.3 mm. A sample to detector distance of 1077 mm gave theq-range 0.07<q<2.9 nm⁻¹. The 2D SAXS patterns were acquired within 1 husing a VÅNTEC-2000 detector (Bruker AXS, Karlsruhe, Germany) with anactive area of 140×140 mm² and a pixel size of 68 μm. The samples weremeasured in 1.5 mm quartz capillaries. The scattering curves wereplotted as a function of intensity, l versus q. Scattering from thecorresponding buffer was subtracted as background from all samples.

Example 2

SAXS measurements—were performed at the cSAXS beamline at PSI (Viligen,Switzerland). After mixing the peptides with coacervation buffer, thecoacervates were equilibrated for at least 1 h and sealed in thin-walledquartz capillaries for SAXS measurements. An X-ray beam with awavelength of 1.11 Å (11.2 keV) was used, with a sample to detectordistance of 2152 mm providing 0.05<q<5 nm⁻¹, where q is the length ofthe scattering vector, defined by q=4π/λ sin(θ/2), λ being thewavelength and θ the scattering angle. The 2D SAXS patterns wereacquired at 12 positions on each capillary in triplets for 1 s (36measurements per sample) using a Pilatus 2M detector (Dectris Ltd,Baden, Switzerland; active area 254×289 mm² with a pixel size of 172×172μm²) and integrated into the one-dimensional scattering function l(q)after inspection for beam damage. No beam damage was observed in allinvestigated samples. The scattering curves were plotted as a functionof intensity, l versus q. Scattering from the corresponding buffer wassubtracted as background from all samples before further analysis.

SAXS Data Analysis—the p(r) was calculated from the scattered intensityl(q) using the following equation (Glatter (1977) J. Appl. Crystallogr.10, 415-421):

$\begin{matrix}{{I(q)} = {4\pi{\int_{0}^{\infty}{{p(r)}\frac{\sin({qr})}{qr}{dr}}}}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

and gives a real space representation of the overall shape of theparticles. The scattering of mass fractal like aggregates was calculatedaccording to Mildner et al. (Meakin et al. (1986) J. Phys. D: Appl.Phys. 19 1535-1545) using SASView.

Prediction of Isoelectric Point of HBP-1 Protein

Isoelectric point (pl) of the HBP-1 protein was predicted usingProtParam tool available online: https://web.expasy.org/protparam/.

Solid-State NMR Spectroscopy

Sample preparation—DgHBP-1 and GY-23 peptide coacervates were loadeddirectly into 1.9 mm MAS rotor by ultracentrifugation (100 k g, 30 min,20° C.) using spiNpack (Giotto Biotech., Italy) rotor packing device.

NMR experiments—NMR data was collected on a 600 MHz Bruker Advance IIIinstrument equipped with a 1.9 mm MAS probe operating in HX doubleresonance mode. One-dimensional (1D) ¹H-¹³C crosspolarization (CP), ¹³Cdirect-polarization (DP) and 2D ¹³C-1³C dipolar assisted rationalresonance (DARR) experiments were performed with the MAS spinningfrequency set at 18 kHz and the variable temperature set at 2° C. Theactual sample temperature was 10° C. based on the external calibrationwith ethyleneglycol (Van Geet, A. L. (1968) Calibration of the methanoland glycol nuclear magnetic resonance thermometers with a staticthermistor probe. Anal. Chem. 40, 2227-2229). Chemical shifts werereferenced using the DSS scale with adamantane as a secondary standardfor ¹³C⁴⁹ (downfield signal at 40.48 ppm) and were calculated indirectlyfor ¹H. The ¹H→¹³C CP transfer was achieved by using 56 kHz ¹³C and 81kHz (maximum power)¹H spin-lock rf fields with a 90%-100% linear rampapplied on the ¹H channel and a contact time of 250 μs. 80 kHz SPINAL-64¹H decoupling was implemented during data acquisition. The recycledelays were 1.5 s and 5 s in the 1D CP and DP experiments, respectively,and the acquisition time was 19.1 ms in both experiments. Additionalparameters of the 2D ¹³C-¹³C DARR experiment included 1.5 s recycledelay, 72115.4 Hz sweep width and 14.2 ms acquisition time in the directdimension, 36000 Hz sweep width and 7.1 ms acquisition time in theindirect dimension and 100 ms DARR mixing time. A dipolar based 2D¹H-¹³C heteronuclear correlation (HETCOR) experiment was conducted with35 kHz MAS rate. The variable temperature was maintained at 15° C.corresponding to 13° C. actual sample temperature. 86 kHz ¹H and 50 kHz(maximum power)¹³C spin spinlock rf fields with a 90%-100% linear rampapplied on the ¹³C channel were implemented for the ¹H→¹³C and ¹³C→¹H CPtransfers and the contact time was 100 μs. Suppression of water signalwas achieved by implementing the MISSISSIPPI scheme without thehomospoil gradient (Morcombe, C. R. & Zilm, K. W. (2003) Chemical shiftreferencing in MAS solid state NMR. J. Magn. Reson. 162, 479-486).Additional parameters of the 2D ¹H-¹³C HETCOR experiment included 1.5 srecycle delay, 34722.2 Hz sweep width and 11.1 ms acquisition time inthe direct dimension, 35000 Hz sweep width and 7.3 ms acquisition timein the indirect dimension, 10 kHz XiX ¹H decoupling during ¹³C chemicalshift evolution period and 10 kHz WALTZ-16 13C decoupling during ¹Hacquisition time.

Isotope Labelling of NMR Samples

¹³C- and ¹⁵N-labeled HBP-1 protein was expressed in E. coli (BL-21 (DE3)strain). A plasmid carrying the protein gene was transformed intochemically competent cells. Bacteria were cultivated in M9 minimalmedium containing ¹³C-glucose (2.5 g/L) and ¹⁵N-ammonium chloride (1g/L), supplemented with biotin (1 mg/L) and thiamin (1 mg/L). Proteinexpression was induced with 0.5 mM IPTG (Isopropylβ-D-1-thiogalactopyranoside) when OD_(600 nm) reached 0.6. The inducedbacterial culture was incubated for 8 h at 37° C. at 225 RPM andharvested by centrifugation (5000 g, 15 min, 4° C.). Cell lysis andprotein purification was carried out as described previously (Tan et al.(2015) Nat. Chem. Biol. 11, 488-495;. Mohammadi et al. (2018) Commun.Biol. doi:10.1038/s42003-018-0090-y).

Uniformly ¹³C and ¹⁵N labelled GY-23 peptide was expressed usingmodified gene of HBP-1-V5 protein variant. The construct was composed ofthe N-terminal part (M1-A112) of the HBP-1 protein, followed by atrypsin recognition site (K), the GY-23 sequence, and a stop codon. Thefusion protein was expressed and purified using the same protocol as for¹³C-¹⁵N-HBP-1 protein. The GY-23 peptide was cleaved off fromHBP-1(M1-A112) tag with trypsin and purified as described above.

Protein Expression and Purification

Genetic constructs encoding wild type HBP-1 and -2 proteins wereobtained from DNA2.0/ATUM (USA), HBP1-V1-C and -V2-C variants wereprovided by Protein Production Platform (School of Biological Sciences,NTU, Singapore), HBP-1-V-3-7 were purchased from GenScript (USA). Theproteins and their variants were expressed in E. coli and purified withreverse-phase HPLC using the protocols developed for the wild type HBP-1and -2 proteins, described previously.

Trypsin Cleavage

The lyophilized HBP-1-V3-7 variants and HBP-2 (wild type) were dissolvedin 50 mM Tris-HCl buffer (pH 8.8) to the final protein concentration of1 mg/mL. Trypsin powder (Trypsin Gold, MS grade, Promega) was dissolvedin 50 mM acetic acid to the final concentration of 1 mg/mL. Enzymaticcleavage was carried out at 37° C. for 2 h at a 1/1000 trypsin/proteinvolume ratio. The reaction was terminated with 1 mM (finalconcentration) PMSF (phenylmethylsulfonyl fluoride). Products of theenzymatic cleavage were purified with reverse-phase HPLC using a21.2×100 mm Kromasil RP-300-C8 column (AkzoNobel, Sweden) and a lineargradient of acetonitrile containing 0.1% TFA (trifluoroacetic acid). Thepurity of separated N- and C-terminal variants was assessed by SDS-PAGEand their molecular weight verified by MALDI-TOF mass spectrometry usingan AXIMA Performance MALDI TOF/TOF Mass Spectrometer (Shimadzu Biotech).Purified products of enzymatic cleavage were freeze-dried andre-solubilized in an appropriate buffer for further studies.

Synthetic Peptides

All unlabeled peptides were synthesized and purified by HPLC (finalpurity >95%) by GL Biochem Ltd (China). GY-23 peptide with ¹³C and ¹⁵Nlabelled tyrosine residues was synthesized and purified using HPLC(final purity >90%) by GenScript (USA).

Encapsulation of Dox and MNPs by Coacervates

Dox (Final concentration: 0.04 mg mL⁻¹) and MNPs (Final concentration:0.008% v/v) were first added to phosphate buffer (pH 9.5 0.1 M) to theirfinal concentrations. DgHBP-2 peptide stock solution (10 mg mL⁻¹) wassuccessively added to the buffer/drug mixture in a 1:9 ratio to triggerencapsulation and coacervation. Gentle pipetting was then done to mixthe final mixture.

Magnetic Alignment of Coacervates Two Nd—Fe—B permanent magnets with thefollowing dimensions (25 mm (length)×12.5 mm (width)×12.5 mm (height)was used to generate uniform magnetic field for alignment ofcoacervates. The distribution and strength of the field was measuredalong ±x and z axis with a gaussmeter (MG-4D, Walker scientific Inc).Magnetic field of ˜200 mT was applied for the alignment of coacervates.

Crosslinking of DgHBP-2 Peptide Coacervates

DgHBP-2 peptide coacervates were first prepared as above. 5 μL of 4-MC(Stock concentration: 100 mM) was added to 50 μL of 1 mg mL⁻¹ DgHBP-2peptide coacervates. The reaction mixture was then diluted to 500 μLwith phosphate buffer (pH 7.5, 0.1M). 5 μL of NaIO₄ (Stockconcentration: 300 mM) was subsequently added and the final mixture wasvortex immediately for 60 s. The crosslinked coacervates were thenincubated at room temperature for 4 hours. After incubation, thecrosslinked coacervates were washed 3× with MilliQ water.

Lysotracker Staining of HepG2 Cells

Liver hepatocellular carcinoma cell line (HepG2) was seeded at 5×10⁴cells per mL into 4 well Lab-Tek chambered glass slide (ThermoFisherScientific) and incubated at 37° C. for 24 hours. After incubation,cells were washed 2× with PBS. GFP loaded coacervates with/withoutcrosslinking were added to the cells with fresh media and incubated at37° C. for 24 hours. The cells were washed 5× with PBS and were addedwith fresh media containing 5 μM Lyostracker Red DND-99 (LifeTechnologies). After 1 hour of incubation, the cells were washed andfixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Thecells were then stained with 2 μg mL⁻¹ of Hoechst 33342 (Sigma Aldrich)in PBS for 15 min, and lastly with 1 μg mL⁻¹ of wheat germ agglutininconjugated to Alexa Fluor 633 (WGA-633 Alexa Fluor) (ThermoFisherScientific) for 10 mins at room temperature. The cells were imaged in aconfocal laser scanning microscope (True Confocal Scanner SP8, Leica).The following excitation (Ex) and emission (Em) wavelength were used:eGFP (Ex: 448 nm, Em: 509); Lysotracker red DND 99 (Ex: 577 nm, Em:590); Hoechst 33342 (Ex: 346 nm, Em: 460); and WGA-633 Alexa Fluor (Ex:632 nm, Em: 590). Micrographs were processed with LAS X software andPhotoshop (Adobe) software.

In Vitro Release Assay (Heat Triggered Dox Release).

50 μL of 1 mg mL⁻¹ of crosslinked coacervates were loaded with 0.04 mgmL⁻¹ of Dox. The coacervates were diluted to a final volume of 200 μLwith 1×PBS and placed in a 2.0 mL Eppendorf tube. The coacervates weresubsequently incubated at respective temperatures for 48 hours. Thecoacervates were centrifuged and the supernatant was analyzed by afluorescence spectrophotometer [Fluorolog, HORIBA Jobin Yvon(Excitation: 470 and Emission: 550)] at different time points. Theremoved supernatant was replaced with new buffer at each centrifugation.

Temperature Measurement of Coacervates Under Alternating Magnetic Field(AMF)

Dox+MNPs crosslinked coacervates were placed inside the copper coils ofAMF generator (Inductelec Limited) with the following magnetic fieldsettings (375 kHz, 1 Amp). Realtime temperature readings of thecoacervates under AMF were taken using fiber optic temperature sensor(Luxtron m600 Temperature Monitor).

Cumulative Release of Dox Overtime

Dox+MNPs crosslinked coacervates were heated in AMF generator(Inductelec Limited) as described above. 100 μL of coacervates weretaken out of the AMF generator at respective timing, followed bycentrifugation and analysis of supernatant by a fluorescencespectrophotometer [Fluorolog, HORIBA Jobin Yvon (Excitation: 470 andEmission: 550)].

Release of Dox by Multiple AMF Treatment

Dox+MNPs crosslinked coacervates were heated by multiple AMF treatments.Each AMF treatment included 20 min heating by AMF generator (InductelecLimited) with 10 mins of cooling. 100 μL of coacervates were taken outof the AMF generator at the end of each treatment and analyzed for Doxrelease using fluorescence spectrophotometer [Fluorolog, HORIBA JobinYvon (Excitation: 470 and Emission: 550)].

Example 1: Glucose-Responsive Peptide Coacervates with HighEncapsulation Efficiency for Controlled Release of Insulin

In this design, the coacervate droplets act as an insulin reservoirwhereas GOx triggers the release of insulin upon exposure to glucose,based on the conversion of glucose to gluconic acid that dissociates thepH-sensitive coacervate droplets and thereby release the insulin cargo(FIGS. 1A-1B).

A twenty-six amino acid long consensus peptide was used derived fromDgHBP-2 (DgHBP-2 peptide) that includes five GHGXY repeats with aC-terminus tryptophan residue (FIG. 2A). To prepare DgHBP-2 peptidecoacervates, buffers of different pH and ionic strength were tested todetermine the optimal conditions for coacervation, which was inferred byturbidimetry. Optimal coacervation occurred between neutral (pH 7.4) toslightly alkaline pH (pH 9.5) (FIG. 2B) and the formation of coacervateswas confirmed by the presence of liquid-like droplets that could beclearly observed by optical microscopy (FIG. 2C). Surprisingly, eventhough ionic strength is usually a key factor governing coacervation(Perry et al. (2014) Polymers 6 (6), 1756; Wang et al. (1999)Macromolecules 32 (21), 7128-7134; Joshi et al. (2018) FoodHydrocolloids 74 (Supplement C), 132-138), further increase in ionicstrength did not greatly affect DgHBP-2 peptide coacervation. SinceDgHBP-2 peptide coacervation did not occur in acidic pH, it was reasonedthat the coacervates may serve as a pH-responsive carrier whose releaseof therapeutic cargo could be triggered by local changes of pH. Throughdynamic light scattering (DLS) measurements (FIG. 3A), the average sizeof DgHBP-2 peptide coacervates was ca. 1 μm and increased by 6% to ca.1.06 μm after insulin encapsulation (FIG. 3B).

Next, it was elected encapsulating insulin within the coacervatedroplets by adding insulin to the phosphate buffer solution. To verifyencapsulation, insulin was labelled with fluorescein isothiocyanate(FITC) to obtain FITC-labelled insulin (FITC-insulin). Followingcoacervation FITC-insulin-loaded coacervates were imaged under aninverted fluorescence microscope. As shown in FIG. 4, the coacervatesand green fluorescence completely overlapped with each other,demonstrating successful encapsulation of insulin by coacervates, withnearly 100% efficiency. Also the cytotoxicity of DgHBP-2 peptide insolution and in the coacervate state on mouse fibroblasts was verifiedusing a live/dead cell assay. As shown in FIGS. 5A-B, the cytotoxicityof DgHBP-2 peptide and insulin-loaded coacervates was negligible at thetested concentrations (FIGS. 5A-B). The encapsulation efficiency (EE)was measured at different insulin/peptide ratios. At 1 mg/mL of peptide,a very high EE was found, reaching nearly 100% regardless ofFITC-insulin concentration (from 0.01 to 0.4 mg/mL) (FIG. 6). The EEslightly dropped only when DgHBP-2 peptide concentration was below 0.3mg/mL (FIG. 7). These results demonstrate the very high loading capacityof DgHBP-2 peptide coacervates, which corroborates previous studiesconducted with other types of coacervates (Johnson, N. R., and Wang, Y.(2014) Coacervate delivery systems for proteins and small moleculedrugs. Expert Opin. Drug Delivery 11 (12), 1829-32). This high EE wasfurther confirmed for labeled-free insulin using reverse phase highperformance liquid chromatography, where native insulin was detectedonly in the coacervates pellet but not in supernatant (FIGS. 8A-D), thusverifying that insulin encapsulation occurs regardless of FITCfunctionalization. The underlying mechanism behind the very high EE ofinsulin within the coacervate microdroplets is still unclear.Nonspecific interactions between DgHBP-2 peptide and insulin are likelyinvolved, notably hydrophobic and π-π interactions given the highcontent of hydrophobic and Tyr residues in DgHBP-2 peptide.

Next, it was tested whether GOx could be co-encapsulated with insulininside the coacervate droplets. As shown in FIGS. 9A-D, the greenfluorescence and the red fluorescence representing FITC-insulin andrhodamine B isothiocyanate-labelled GOx (RITC-Gox), respectively, fullyoverlapped and yielded yellow fluorescence (FIGS. 9A and B). Theseresults established that both insulin and GOx could be co-encapsulatedby DgHBP-2 peptide coacervates with a high efficiency (FIG. 9D).

To investigate whether insulin release could be triggered from thecoacervates by glucose-sensing, GOx+insulin-co-loaded coacervates (1mg/mL of DgHBP-2 peptide, 0.01 mg/mL of GOx and 0.1 mg/mL of insulin) aswell as insulin-loaded (1 mg/mL of DgHBP-2 peptide, 0.1 mg/mL ofinsulin) coacervates (free of Gox) in phosphate buffer were prepared andthe coacervate droplets were exposed to glucose. As shown in FIG. 10A,the insulin release rate was much faster in the presence of both GOx andglucose (FIG. 10A), indicating that glucose readily diffused into thecoacervates and converted to gluconic acid by GOx, resulting in localacidification that dissociated the coacervates.

Although some leakage of insulin was observed in the absence of glucose,the rate and the degree of insulin release over 48 hours weresubstantially higher in the presence of glucose.

Next, the ability of GOx+insulin coacervates was examined to respond tochanges in glucose concentration. The coacervates displayed a pulsatilerelease of insulin when the glucose concentration was altered betweennormal (1 mg/mL) and hyperglycemic levels (4 mg/mL) every 1.5 hours(FIG. 10B). The coacervates could reversibly vary the rate of insulinrelease between higher and slower rates in response to hyperglycemic andnormal levels. Such a glucose-triggered insulin release behavior couldbe repeated for at least 3 cycles.

An important parameter to ensure that this GRIDS system could be usedfor glucose management is to verify retention of insulin activityfollowing entrapment within the coacervate droplets (Blocher et al.(2017) Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 9 (4), e1442)and subsequent release from the local acidic microenvironment. First,Circular Dichroism (CD) measurements were used to assess possiblechanges in secondary structure (FIG. 11A). CD spectra of the regularinsulin solution displayed the classical α-helical signature (two minimaat 208 and 222 nm). After coacervation and release, the CD signature wasa mix of insulin and DgHBP-2 peptide, with the slight decrease inintensity attributed to the weaker ellipticity of the peptide. However,the characteristic minima of α-helices at 208 and 222 nm were stillvisible. In addition, CD spectra of native insulin at pH 5.5 to 7.5(FIG. 12) indicated that the secondary structure was not disrupted atmild acidic conditions, strongly suggesting that released insulinretained its bioactivity. The CD spectrum of thermally denatured insulinwas also measured (FIG. 11A), which exhibited important differences withnative/released insulin, specifically the loss of the 195 nm maximum andof the 222 nm minimum, as well as a peak shift of the 208 nm minimumtowards lower wavelength. To further verify insulin activity, theinsulin-DgHBP-2 peptide solution was subjected after release to an ELISAassay. The released insulin was detected to similar levels compared tonative insulin (FIG. 11B), suggesting that the conformational epitopewas not disrupted since antibodies could equally recognize thecoacervates-released insulin. Together, these data are in agreement withrecent studies showing that therapeutic biomacromolecules retain or evenexhibit enhanced bioactivity inside the coacervate phase (Blocher et al.(2017) Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 9 (4), e1442;Black (2014) ACS Macro. Lett. 3 (10), 1088-1091; Martin et al. (2016)Langmuir 32 (23), 5881-9; Pippa et al. (2015) Int. J. Pharm. 491 (1-2),136-43), and that folded proteins may be sequestrated within coacervatemicrodroplets when compared to their unfolded state (Martin et al.(2016) Langmuir 32 (23), 5881-9.).

In summary, a simple and efficient GRIDS based on biomimetic peptidecoacervates loaded with GOx and insulin was synthesized. The GRIDS canbe easily prepared by simply mixing biomimetic DgHBP-2 peptide intoaqueous buffer solutions containing insulin and GOx. Coacervation ensuesand acts as an insulin reservoir with very high encapsulationefficiency. Under hyperglycemic conditions, glucose molecules diffuseinto the coacervates where GOx readily converts glucose into gluconicacid, resulting in a local acidic environment that triggers thedissociation of coacervates and eventually leads to glucose-triggeredrelease of insulin, with a release kinetics that can be altered inresponse to glucose levels. Furthermore, the secondary structure and theactivity of insulin are retained following exposure to acidicmicroenvironment, which can primarily be attributed to the use ofaqueous solvents. Our results indicate that this new GRIDS combine theadvantages of both glucose responsiveness and high loading capacity,representing a promising potential for diabetes management. Furtheroptimization to enhance coacervate stability is currently underway,notably to prevent premature leakage in the absence of glucose exposure,which may be achieved by introducing simple mutations in the peptidesequence or by varying the number of tandem pentapeptides.

Encapsulation of Other Therapeutics Agents in DgHBP-2 PeptideCoacervates

Other than insulin and GOx, DgHBP-2 peptide coacervates can encapsulatea wide range of therapeutic or functional biomacromolecules. Forexample, it can efficiently encapsulate proteins such as Bovine SerumAlbumin (BSA) and Green fluorescence protein (GFP) (FIGS. 13 and 14).

In addition, DgHBP-2 peptide coacervates can also be loaded withmagnetic nanoparticles (MNP) as well as low molecular weight drugs likeDoxorubicin (DOX) (FIG. 15). These DOX+MNP loaded coacervates can bemanipulated using magnetic fields, opening up the possibility ofmagnetic targeting and magnetic field induced drug release forchemotherapy (see also Example 3).

Family of DgHBP-2 with Coacervation Capability

The ability to self-coacervate is not limited to the peptide describedabove, but can be expanded to a family of Dg-HBP peptides sharing commonsequence design and motifs. In a non-limiting embodiment,stimuli-responsive coacervates for therapeutics release may also beachieved with the following family of peptides:

(GHGXY)_(n)  Sub-family 1:

where X can be V, L, or P, and n is the number of repeats, with is n≥4.

[(GHGXY)_(n)(GAGFA)_(m)]_(i)GHGXY  Sub-family 2:

where X can be V, L, or P, n is the number of repeats of sub-peptide 1GHGXY (with n≥1); m is the number of repeats of sub-peptide 2 GAGFA(with m≥1), and i≥2.

[(GHGXY)_(n)(GHGLH)_(m)]_(i)GHGXY  Sub-family 3:

where X can be V, L, or P, n is the number of repeats of sub-peptide 1GHGXY (with n≥1); m is the number of repeats of sub-peptide 2 GHGLH(with m≥1), and i≥2.

Examples for each sub-family that have been successfully tested forself-coacervation (using turbidity measurements and observation ofdroplets by optical microscopy) include the following:

For sub-family 1:  (SEQ ID NO: 15) GHGVY GHGVY GHGPY GHGPY GHGLYFor sub-family 2:  (SEQ ID NO: 16) GHGLY GAGFA GHGLY GAGFA GHGLYFor sub-family 3: (SEQ ID NO: 17) GHGLY GHGLH GHGLH GHGLH GHGLY

Example 2: Aromatic Interactions within Repeat Motifs TriggerpH-Responsive Liquid-Liquid Phase Separation of Intrinsically DisorderedPeptides

Mutagenesis studies with both solution- and solid-state NMR spectroscopywere combined to investigate the coacervation process of DgHBP-1. Acentral characteristic of DgHBPs is the presence of repetitive regionsin their C-termini that share similarity with the hydrophobic repeats oftropoelastin (Cai et al. (2017) Soft Matter 13, 7740-7752). Thus,DgHBP-1 was selected as a model structural IDP to shed light on sequencemotifs that govern LLPS as well as on intermolecular interactionsstabilizing the coacervate phase. The DgHBP-1 sequence wassystematically explored and it was identified that the motif repeatGHGLY drives LLPS. It was demonstrated that at least two copies of suchrepeats and a linker sequence must be included, or alternatively atleast four tandem repeats must be present in order to triggercoacervation. Within this motif it was shown that His residues serve asa molecular switch: upon pH change, they first undergo deprotonationfollowed by hydrogen bonding with Tyr. Finally, using solution-,solid-state NMR, and Small Angle X-ray Scattering (SAXS) it wasdemonstrated that clustering of Tyr residues is critical to stabilizecoacervate micro-droplets.

DgHBP-1 is Structurally Disordered in Solution

DgHBP-1 possesses primary structure features characteristic of IDPs withLLPS properties (FIG. 16). In a recent study, it was shown usingCircular Dichroism (CD) and SAXS that it has a dynamic, disorderedmolecular structure in solution that transitions to a more ordered formin the coacervate state, and it was proposed that hydrophobic modularpenta-repeats from the C-terminus are key to the coacervation process(Cai et al. (2017) Soft Matter 13, 7740-7752). To verify theseassumptions and investigate the structural features of the protein, astandard set of double- and triple-resonance NMR experiments withsoluble recombinant DgHBP-1 was carried out. As predicted, NMR resultsindicated that the protein lacked a defined three-dimensional structurein solution: the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC)spectrum (FIG. 17A) showed narrow distribution of the amide protonchemical shifts, which is typically observed for IDPs with LLPSproperties (Conicella et al. (2016) Structure 24, 1537-1549; Brady etal. (2017) Proc. Natl. Acad. Sci. 201706197.doi:10.1073/pnas.1706197114; Burke et al. (2015) Mol. Cell 60, 231-241).Analysis of Ca and CP chemical shifts of assigned residues did not showsignificant deviations from random coil values, validating that themonomeric DgHBPs are uniformly disordered (FIG. 18).

Residues Located at C-Terminus of DgHBPs are Involved in the pHDependent LLPS

LLPS of DgHBPs is triggered by changes in pH and ionic strength. DgHBP-1underwent LLPS at a minimal concentration of 20-30 μM in a narrow pHrange 6.5-7.5, which is close to the proteins' isoelectric point(predicted pl=6.03) and could be broadened by increasing protein andsalt concentration (phase diagrams presented in FIG. 19). To preciselyprobe the residues involved in LLPS of HBP-1, a set of ¹H-¹⁵N-HSQCspectra with a gradual increase of the pH from 3.3 (soluble state) (FIG.17A) to 6.5 (at which point LLPS was initiated (FIGS. 20A-E)) wasrecorded. Finally, the spectrum from the diluted phase after LLPS wasmeasured, when the coacervate micro-droplets had sedimented (FIG. 17B).The overlay with the spectrum acquired in initial conditions (FIG. 17C)indicated the absence of resonances assigned to glycine (Gly), His,alanine (Ala), and leucine (Leu) residues located mainly in theC-terminal modular repetitive region, suggesting that these residueswere involved in transient interactions that were absent at acidic pH.As a control a set of spectra at 75% lower concentration compared to theinitial conditions (FIGS. 21A-D) and at lower temperature (279° K vs.298° K in initial conditions, FIGS. 22A-D) was acquired to probepossible exchange between monomeric and oligomeric states or exchangewith water molecules, respectively. For both experiments at pH 6.5, theintensity losses of the same cross-peaks were detected, confirming thespecific involvement of these residues (located mostly in the modularrepeats of DgHBP-1) during LLPS.

Analysis of Modular Repeats Driving Phase Separation of DgHBPs—Key Roleof GHGxY Motifs

To study how the C-terminal modular domains' arrangement influencesself-coacervation of HBP-1, a series of sequence variants was designed(FIG. 23A-D, full sequences in FIGS. 24A-C; SEQ ID Nos. provided inFigure description) and their ability to phase separate at various pHand salt (NaCl) concentration was investigated using optical microscopy(FIGS. 23E-F). First, a protein mutant lacking the first 66 amino acidsbut containing all modular repeats of the C-terminus (V1-C) was created.This mutant underwent phase separation and formed coacervates at similarprotein concentration and pH range compared to the full-length protein,confirming hypothesis that C-terminal modular repeats are responsiblefor its phase separation behavior. Next, a variant lacking the first 31amino acids of the repetitive region (V2-C) was studied. This variantformed coacervates similarly to V1-C and DhHBP-1 wild type but requireda slightly higher protein concentration (ca. 30 μmol), indicating thatthe full length of the modular region was not required to induce phaseseparation.

To map out the minimal sequence length required for phase separation, aseries of DgHBP-1 mutants with various lengths of the repetitive regionwas designed. The mutants were created by introducing a single Lys atdifferent pre-selected locations, allowing to utilize trypsin cleavageto tune the length of the cleaved fragments following enzyme digestionas well as to obtain variants exhibiting different lengths of therepeating domains (FIG. 23C and FIG. 24B).

Then the LLPS behavior of all variants as a function of proteinconcentration and pH was analyzed, and at various salt concentrationsand drew the phase diagrams shown in FIG. 23E. For N-variants, LLPSoccurred for V5-N to V7-N only at high salt concentrations. On the otherhand, LLPS could not be induced for V3-N and V4-N at all testedconditions. It was also observed that as peptide length increased, LLPSoccurred over a broader range of conditions. Thus, for V7-N LLPS couldbe induced at pH as high as 8 provided the peptide concentration was atleast 500 μM. For V6-N, the highest pH at which LLPS was observed was 7(and a minimal peptide concentration of 400 μM), whereas for V5-N noLLPS occurred above pH 6. Correlating the results with the peptidedesign point out towards the importance of the GHGLY motif and thepeptide length. For the longer V6-N and V7-N peptides containing twoGHGLY motifs, LLPS could be induced over a wider range of conditions,whereas for the shorter V3-N and V4-N variants containing only one copyof GHGLY, no LLPS was observed no matter the conditions. And for theintermediate length V5-N with one GHGLY motif, LLPS could be induced butonly under narrow conditions. Moreover, the separated phases of thelonger variants exhibited a different morphology compared to thefull-length protein (FIG. 23F), forming dense hydrogel-like structuresthat did not disperse into the surrounding buffer. This behavior may belinked to the stronger hydrophobicity of V5 to 7-N compared to othervariants, which may favor hydrogel formation by hydrophobicinteractions.

A similar trend was observed for the C-terminus variants. V3-C, whichcontained the longest section of the repetitive region, phase-separatedat the lowest protein concentration (30 μM at pH 8) and in the broadestpH range among all tested variants. On the other hand, the shorter Vx-Cvariants exhibited LLPS under a narrower range of conditions andrequired higher protein concentrations.

To further assess the role of the GHGLY motif, the coacervation abilityof the DgHBP-1 derived GY-23 peptide (containing two GHGLY copies) wascompared with two other synthetic peptides made of very similarfragments of HBP-1 repeats (GA-25 and GH-25), but harboring only oneGHGLY motif (FIGS. 23D and 24C). Only GY-23 phase-separated, formingcoacervate micro-droplets suspended in solution as well as a densehydrogel-like structure (condensed, solid-like coacervates, FIG. 23F).In contrast, GA-25 and GH-25 remained in solution in all tested bufferconditions (FIG. 23F). It should be noted that sequence motifs similarto GHGLY are also present in the C-terminal of DgHBP-2 protein, whichcontains seven copies of the GHGxY motif (where x can be Val, Pro, Leu)arranged in tandem (FIGS. 25A-C). A peptide (DgHBP-2-pep) composed of 5copies of GHGxY was previously shown to phase separate and formcoacervates in the same way as the full-length protein.

In order to confirm the central role of GHGxY motifs on LLPS of DgHBP-2,trypsin cleavage was utilized to obtain shorter fragments of DgHBP-2 andtheir ability to phase-separate was tested. Since the protein possessesonly two trypsin recognition sites at positions R81 and R172, weobtained the N-terminal (M1-R81) fragment that lacked the modularrepeats, the C-terminal (λ82-R172) containing the whole repetitiveregion, and a short G173-Y175 peptide that was discarded (FIG. 25B). Asexpected only the C-terminal fragment phase-separated into coacervates(FIG. 25C). Next, a series of short peptides containing differentarrangement of repetitive units present in DgHBP-1 and DgHBP-2 (FIG.26A) we designed and their phase separation behavior was analyzed in thesame way as for DgHBP-1 variants. Phase separation was observed for all25-mer peptides containing two GHGLY motifs flanking the central regioncomposed of 3 copies of the GAGFA or GHGLH sequences, as well as for a20-mer peptide (GY-20) made of four copies of GHGLY motif arranged astandem repeats. In contrast, no phase separation was observed when thepeptide length was reduced to 15 amino acids, for example when 3 copiesof the GHGLY motif were arranged in tandem (GY-15-V1) or when the GAGFAmotif was flanked by GHGLY (GY-15-V2). Similarly, no phase separationwas observed for decapeptides composed of one or two GHGxY motifs or forpentapeptides GHGLY or GAGFA, respectively. Moreover, peptides with LLPSability exhibited various rheological characteristics of the separatedphase. GY-25-V1 peptide containing 3 copies of hydrophobic GAGFA motifphase-separated into a dense and compact hydrogel. On the other hand,GY-25-V2 and GY-20 peptides composed of less hydrophobic, His-richmotifs, only formed micro-droplets (FIG. 26B), while GY-23 peptidecontaining both types of motifs separated into micro-droplets as well ashydrogel-like condensed coacervates (FIG. 23F).

Taken together these results indicate that when at least two copies ofthe GHGLY motif are present in the tandem repeats, the phase separationability is greatly enhanced. However, this condition is not sufficientand GHGLY copies must additionally be separated by a spacer composed ofat least 3 copies of GAGFA or GHGLH motifs, or a combination ofGAGFA/GFA and GHGLH motifs. Alternatively, the peptide must contain atleast four tandem repeats of GHGLY motif to phase-separate. Tocorroborate the role of Tyr in phase separation, two GY-23 variants inwhich one of two Tyr was substituted with Ala (FIG. 26D) were prepared.Phase separation did not occur in both cases and in all testedconditions, further pointing out to the critical role of Tyr-Tyrinteractions in driving phase separation.

Modular Interactions Initiating LLPS—Solution-State NMR Spectroscopy

Subsequently, NMR spectroscopy studies were carried out to assess therole of Tyr residues and to identify the detailed molecular interactionstriggering and stabilizing LLPS. First, the ¹H-¹⁵N-HMQC spectrum insolution as well as a set of triple-resonance NMR spectra for peptidebackbone assignment of soluble GY-23 at pH 3.3 was acquired. The¹H-¹⁵N-HMQC spectrum yielded well-resolved peaks that could be fullyassigned based on the carbon chemical shifts values obtained from the 3Dexperiments (FIG. 27A). Observed Ca and CP chemical shifts showed nosignificant differences from the average values of random coilstructures (FIG. 28), confirming that the peptide displayed nopropensity towards a specific secondary structure.

Next, the pH of the peptide solution has been titrated and changes inthe ¹H-¹⁵N-HMQC have been monitored (FIG. 27B). Major variations in thepeak distribution and relative intensity at pH 4-6 were not observed(FIG. 29) compared to the initial state (pH 3.3, FIG. 30A), since inthese conditions the peptide remained fully soluble. However, close tothe LLPS point between pH 6-7, there was an observable major shift anddecrease in the relative intensity of all cross-peaks assigned to Hisresidues (FIG. 29) as well shifts in the positions of all Gly peaksflanking them. In addition, shift of the cross-peak assigned to Y5 wasobserved (FIG. 27B).

These results indicate that His and Tyr residues may be involved ininitiating LLPS. To investigate their

role in initial steps of aggregation, pH titration experiments on GY-23have been carried out, where ¹H-¹³C-HSQC spectra of aliphatic (FIG. 27C)and aromatic (FIG. 27D) side chains of all residues were recorded, aswell as the long range ¹H-¹⁵N-HMQC spectrum to monitor the protonationstate of nitrogen atoms in the imidazole ring of His (FIGS. 27E-F andFIGS. 31A-B). Increasing the pH led to gradual changes of chemicalshifts of His ¹³C_(α) and ¹³C_(β) atoms (upfield in the ¹H dimension anddownfield in the ¹³C dimension), as well as ¹³C_(δ) and ¹³C_(ε) atoms ofthe imidazole ring (FIG. 27D). In addition, resonances assigned to1³C_(α) and ¹³C_(β) of C-terminal Y23 residue significantly shiftedupfield in the ¹H dimension and downfield in the ¹³C dimension when thepH raised from 3 to 4, suggesting that the shift is caused bydeprotonation of the C-terminal carboxylic group. A major shift of the1³C_(α) cross-peak assigned to G1 was observed as well (FIG. 27C).

¹H-¹³C-HSQC spectra of aromatic side chains showed that increasing pHresulted in gradual shifts of resonances assigned to ¹³C_(δ2) and1³C_(ε1) of His residues, which is related to deprotonation of theimidazole

ring. Resonances assigned to Tyr and Phe remained unaffected by pHchange between 3.3 and 6. However, when the pH increased from 6 to 7 asplit of Tyr ¹³C_(δ) resonances into two peaks and a minor upfield shiftin ¹H-dimension of all Phe resonances were observed. In addition,chemical shifts of ¹³C_(ζ) atoms of Y5 and Y23 at pH 7 were split (FIG.27D, inset), suggesting that they were in different chemicalenvironments. FIG. 27E shows changes in chemical shifts of ¹⁵N atoms ofHis imidazole ring during pH titration. At pH 3.3 and 4 all His werefully protonated (since the ¹⁵N_(ε2) and ¹⁵N_(δ1) exhibited typicalchemical shift values for the protonated state, i.e. 173 ppm and 176ppm, respectively) (Pelton, J. G., Torchia, D. A., Meadow, N. D. &Roseman, S. (1993) Tautomeric states of the active-site histidines ofphosphorylated and unphosphorylated IIIGlc, a signal-transducing proteinfrom Escherichia coli, using two-dimensional heteronuclear NMRtechniques. Protein Sci. 2, 543-558). Increasing the pH from 4 to 7 ledto the gradual deprotonation of the imidazole rings of all His andco-existence of the fully protonated state with two tautomeric forms ofthe imidazole ring. Critically, it was observed that immediately afterraising the pH from 6 to 7, only one of four His residues showedtransient stabilization of its E tautomer state since the ¹⁵N_(δ1) peakappeared also at 250 ppm within 5 min after pH adjustment (FIG. 27F).However, the cross-peak intensity was significantly reduced 30 minfollowing pH adjustment (FIGS. 31A,B), indicating that only one Hisresidue underwent transient stabilization of the tautomeric state, whichis likely caused by hydrogen bonding. Since at pH 7 chemical shifts ofTyr ¹³CC atoms were also shifted, this suggests that hydrogen bondinteraction is taking place between the hydroxyl group of Tyr and¹⁵N_(δ1) of His, which may be the first step in the oligomerizationcascade. Moreover, 3D ¹⁵N- and ¹³C-NOESY experiments with long mixingtimes were carried out and NOEs between His and Tyr were not observed,further supporting the transient character of the Tyr/His interaction.GY-23 Peptide Shows Partially Ordered Structures after LLPS

Although IDPs do not exhibit well-defined tertiary structures, there areevidences that coacervate micro-droplets of IDPs contain short-rangeorder¹⁰. To further study the coacervation at the nanostructural leveland assess whether GY-23 coacervate micro-droplets exhibited suchinternal ordering, we investigated their structural features using SmallAngle X-ray Scattering (SAXS). Scattering profiles of GY-23 in aceticacid (pH 3.3) before LLPS and in the coacervate buffer (pH 7.0) afterLLPS (both the coacervate and the coexisting dilute phases) arepresented in FIG. 30A and were very distinct from each other. Thescattering intensity of GY-23 in acetic acid and of the dilute phaseafter centrifugation had a very low signal-to-noise ratio. Nevertheless,for GY-23 in acetic acid, a weak low-q upturn with an indication of abroad correlation peak between 0.3 and 2 nm⁻¹ was observed, which may beattributed to nanometer-sized peptide oligomers. Dynamic lightscattering (DLS) analysis of the peptide in acetic acid (FIG. 30C)indicated the presence of structures with a hydrodynamic diameter(D_(H)) of Ca. 8 nm, corroborating the presence of small oligomericunits (assuming D_(H) on the order of 4-8 nm for the 23 residue-longmonomeric peptide). As expected, D_(H)increased drastically to around 50nm at pH 7.0 due to initiation of LLPS.

In contrast, the scattering profile of GY-23 in the coacervate phase(FIG. 30A) indicated the presence of much larger peptide aggregates inthe form of coacervate micro-droplets with overall dimensions thatexceeded the resolution limit of the SAXS set-up. An indication of abroad correlation peak in the q-region of ˜1.5 nm⁻¹ suggested structuralfeatures from peptide self-assemblies within the coacervatemicro-droplets. The low signal-to-noise ratio in this q-region makes itdifficult to analyze this feature in detail (however this correlationpeak was confirmed using a more intense synchrotron x-ray source, FIGS.32A-B). At q<1 nm⁻¹, on the other hand, the scattering curve showed anapproximate power-law dependence over at least an order of magnitude inthe q-range, indicating fractal scattering from the dense peptideassemblies within the coacervate phase.

To further investigate the internal structure of coacervatemicro-droplets, the pair distance distribution function p(r) wascalculated from the SAXS curve using the indirect Fourier transformation(IFT) method (FIG. 30B). The p(r) function reflected large peptideaggregates in the micro-droplets with dimensions well-beyond theresolution limit of the SAXS set-up in this study (around 50 nm in realspace). Hence, the p(r) was mathematically forced to 0 at r around 100nm, but this does not represent the overall dimension of the coacervatemicro-droplets. The analysis of the corresponding SAXS data of thecoacervate droplets in buffer at a higher signal-to-noise ratio,recorded at the synchrotron, is presented in the FIGS. 32A-B. Theresults indicated that the coacervates micro-droplets containednanostructural features of Ca. 2 nm. These features are most likelyattributed to oligomeric peptides forming the internal domain structuresof the coacervate micro-droplets.

Analysis of Tyrosine-Tyrosine Interactions by ssNMR

Since site-directed mutagenesis experiments suggested a critical role ofTyr residues, GY-23 containing uniformly labeled (¹³C and ¹⁵N) Tyrresidues (Y5 and Y23) were synthesized and possible Tyr-Tyr interactionsin the condensed coacervate phase by solid-state NMR were analyzed.FIGS. 33A-B show a comparison between 1D direct and ¹H-¹³C CP-basedcarbon spectra. Both spectra contained relatively broad lines,indicating that Try residues were present in heterogeneousconformational environments since multiple peaks for each Tyr carbonwere observed. For example, ¹³C_(α) resonances at 53.0, 57.5, 58.7 ppm,and carbonyl ¹³C at 173.3, 176.8, 180.8 ppm, respectively, weredetected. The presence of strong signals in CP-based spectrum indicatedthat most of Tyr moieties were locked in the rigid structure with highdipolar order. No extra sharp peak was observed in thedirect-polarization ¹³C spectrum compared with the CP-based spectrum,indicating the absence of highly flexible Tyr residue, hence furthersupporting that Try residues were rigidly locked. Two-dimensional¹³C-¹³C Dipolar Assisted Rotational Resonance (DARR) (FIG. 33C) showscorrelations between the two Tyr residues of the peptide, demonstratingthat they interacted with each other. Moreover, the DARR data clearlyindicated that Tyr residues were in heterogeneous chemical environments,implying clustering of Tyr residues close to each other. Tyr-Tyr directinteractions were also corroborated by the heteronuclear correlation(HETCOR) spectrum (FIG. 33D), which shows correlations between aliphaticand aromatic carbon atoms of Tyr attributed to the stacked clustering oftwo or more tyrosine side groups.

Example 3: Doxorubicin (Dox)-Loaded Magnetic Coacervates forChemotherapy Dox-Loaded Magnetic Coacervates

Dox and MNP are first added to buffer, followed by additional of peptidestock solution to initiate coacervation and encapsulation (FIG. 36A).

Dox+MNP loaded coacervates can be directed away from health tissue andaccumulated at tumour site using directional magnetic field. Once thecoacervates reach the target site, heat generated from MNP underalternating magnetic field (magnetic hyperthermia) induces the releaseof Dox. Heat generated can also enhance the cytotoxic activity of Dox(FIG. 36B).

Heat Dissociation of Coacervates

One of the best known proteins that can be used for coacervates istropoelastin. However, tropoelastin is not suitable in this case becauseit requires heat to drive coacervation. The level of tropoelastincoacervation will increase with magnetic hyperthermia, making release oftherapeutics difficult.

DgHBP-2 peptide coacervates dissociate upon heating. Therapeutics thatare loaded inside DgHBP-2 peptide coacervates are released, which isstimulated by magnetic hyperthermia. By controlling the duration ofmagnetic hyperthermia, the rate of therapeutics release can becontrolled (FIG. 37).

Dox+MNP Loaded Coacervates

Dox has a natural red fluorescence, allowing the detection of Dox underfluorescence microscopy. From the microscopy images, the co-localisationof red fluorescence and droplets-like coacervates indicated that Dox hasbeen encapsulated successfully (FIG. 38).

Without magnetic field, Dox+MNP loaded coacervates were randomlydeposited to the glass slides. Upon exposure to magnetic field, thecoacervates lined themselves along the magnetic field and formed stringsof coacervates (FIG. 38).

Encapsulation Efficiency of Different Concentrations of Dox byDgHBP-2-Peptide Coacervates

Only ˜40% of Dox is being encapsulated (FIG. 39), thereforeunencapsulated Dox (60%) need to wash off. However, once coacervates arecentrifuged, they cannot be retrieved (stick to Eppendorf tube aftercentrifugation). To wash off excess Dox, coacervates have to be firststabilised. Stabilisation can be done by crosslinking the coacervates.

One possible reason for the difference in encapsulation efficiency ofinsulin (>99%) and Dox (˜40) could be due to charge difference. Insulinhas an isoelectric point of 5.4 and is negatively charged atphysiological pH. Dox has a pKa value of ˜8.46 and is positively chargeat physiological pH. However, the effect of charge on encapsulation incoacervates is currently unknown and remains to be verified.

Covalent Crosslinking of Catechol

Catechols can be oxidised to quinones for covalent crosslinking. Thereare two main methods of oxidation: chemically and enzymatically. Acommon chemical oxidant, sodium periodate (NaIO₄) was used in thisproject to convert catechols to quinones (FIG. 40(https://bioinspiredmaterials.berkeley.edu/research/mussel-inspired-adhesives/)).Quinone are extremely reactive and can react with different chemicalgroups in the peptide e.g. amines and imidazoles to form covalent bonds.

4-Methylcatechol (MC)/NaIO₄ Crosslinked Coacervates

The co-localisation of red fluorescence and 4-MC/NaIO₄ coacervatesindicated that Dox still retains within the coacervates aftercrosslinking. Under a directional magnetic field, the 4-MC/NaIO₄coacervates lined themselves along the magnetic field and formed stringsof coacervates. These results show that by using a magnetic field, thecoacervates can be directed towards tumour site or to accumulate attumour site and away from healthy tissue. The level of crosslinking canbe fine tuned between stabilisation and Dox release by changing4-methylcatechol concentration (FIGS. 41A-D).

Heat Induced Release of Dox

The rate of Dox release at different temperatures from 4-MC/NaIO₄crosslinked coacervates was measured. Rate of Dox release at 37° C. wasmeasured to determine the leakiness of coacervates in normal bodytemperature. Rate of Dox release at 42° C. and 45° C. was assessed tosimulate magnetic hyperthermia temperatures. From the data, the rate ofrelease at 42° C. and 45° C. is much higher compared to 25° C. and 37°C. This confirms that release of Dox can be triggered by heating frommagnetic hyperthermia. Without magnetic hyperthermia, the release of Doxis minimum (FIGS. 42A-B).

Cell Viability Assay (MTT Assay)

NaIO₄ and 4-MC are toxic chemicals that may be harmful to cells. Aftercrosslinking, excess unreacted chemicals must be washed off. MTT assaywas used to test for toxicity of crosslinked coacervates and remnants oftoxic chemicals. The results reveal that the crosslinked coacervates byitself were nontoxic and excess chemicals have been removed aftercrosslinking (FIG. 43A). This shows that Dox leakage from coacervates isminimum and does not affect the viability of cells (FIG. 43B) (MTT:(3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)).

Dox loaded coacervates were incubated with HEPG2 liver cancer cells toassess its cytotoxic properties. FIG. 44A: Dox-loaded coacervates in PBSwithout heat treatment, FIG. 44B: Dox-loaded coacervates in PBS wereheated at 45° C. for 30 min, before adding to cells (measurement at 570nm). This heating is to induce the release of Dox. The relative cellviability of non-heated coacervates treated cells is more than 80%,whereas relative cell viability of heated coacervates treated cellsdecrease to -50%. This indicates that Dox still retains its cytotoxicproperties after its encapsulation and release from coacervates.Relative cell viability of heated coacervates treated cells increases asthe concentration of coacervates decreases. This is because as theconcentration of released Dox decreases, the cytotoxic properties of Doxalso decrease.

GFP Loaded Coacervates

Despite the size of coacervates (˜1 μm), they were able to enter HepG2cells within 3 hours and remained stable inside the cells for at least24 hours. The coacervates were not broken down in lysosomes or by otherenzymes present in cytosol. They were also unlikely to be trapped inorganelles like endosomes, as coacervates were still able tocoalescence. The stability of coacervates inside the cells implies thatany Dox will remain encapsulated until magnetic hyperthermia occurs. Therelease of Dox can be controlled in a more precise manner (FIGS. 45A-C).

Endosomes Staining (Endocytosis)

Endocytosis is a kind of active cell uptake process where particles e.g.parts of cells, or nutrients are taken into cell. During thisinternalisation process, cell membrane invagination occurs to form amembrane-bound compartment called endosomes. Since endocytosis is one ofthe main cell uptake processes, endosomes staining is done to determineif coacervates are inside endosomes after cell uptake. Lysotracker is adye that stains acidified endosomes. Green fluorescence of GFP does notco-localize with the red fluorescence of Lysotracker. These resultsshowed that coacervates are not in the endosomes, therefore they areunlikely to enter through endocytosis. Another possibility is that thecoacervates enter cells through endocytosis pathway, but escape from theendosomes before the latter fuses with lysosomes. The orthographic viewsreveal that coacervates were inside cells and not sticking to the cellmembrane (FIG. 46).

The GFP-loaded coacervates were located between the cell membrane andnucleus, indicating that the coacervates had successfully entered thecell (3D image GFP-loaded coacervates treated HEPG2 cells, FIG. 47).

Heating of Dox+MNP Loaded Coacervates by Alternating Magnetic Field(AMF)

Under alternating magnetic field, MNP and Dox+MNP loaded coacervateswere heated to more than 50° C., whereas PBS (without any MNP) washeated to only 38° C. (FIG. 48). This slight increase in temperature forPBS was due to the heat generated from the AMF generator.

MHT Triggered Release of Dox

Dox+MNP loaded, 4-MC/NaIO₄ coacervates were placed in alternatingmagnetic field to induce magnetic hyperthermia. The heat from magnetichyperthermia is used to trigger the release of Dox. From the data, theamount of Dox released increases with the duration of magnetichyperthermia. At 40 min, ˜25% of Dox in coacervates were released. Sincenot all the Dox is released, multiple treatments can be done with singleinjection (FIGS. 49A-B).

Preliminary Animal Studies In Vivo Work-Mouse (Biodistribution)

Free cy5.5 dye is clear mainly through kidneys. Since highest radiantwas observed in liver, this suggests that the coacervates are stable invivo as the cy5.5 dye still remained inside the coacervates. The rapidclearance of coacervates by liver can be due to the size of coacervates(BL6 female mice: FIGS. 50A, B, C, apolipoprotein E knockout (APOE KO)mouse: FIGS. 50D and E). The APOE KO mice do not have apolipoprotein Ein their body and are more susceptible to plaque formation in theiraorta. The first set of mouse work data (FIGS. 49A and 49B) usedwildtype mice whereas the second set of mouse work (FIGS. 49D and 49E)used APOE KO mice. The reason for switch in the type of mice used was totest if the coacervates could attached to the plaques for prospectiveplaque-targeted delivery therapies.

There has been growing recognition that LLPS is involved inside cellsvia membrane-less organelles as well as in the processing ofextra-cellular load bearing structures and bioadhesives of variousorganisms. However, sequence motifs and associated inter- andintra-molecular interactions driving phase separation remain sparselyunderstood. It was shown that phase separation of DgHBPs is mediatedthough specific GHGXY modular repeats that must be arranged in aspecific configuration. It was also shown that the morphology andrheology of separated phases can be tuned from dispersed coacervatemicro-droplets to hydrogels by incorporating hydrophobic GAGFA repeatsin a peptide sequence. Based on solution-state NMR measurements, LLPS ofDgHBPs is a multi-step process initially triggered by deprotonation ofHis residues upon pH increase, followed by stabilization of His Etautomeric state by transient hydrogen bonding with OH group of Tyrresidues. Without wishing to be bound to any particular hypothesis, itis believed that these events eventually promote hydrophobicinter-molecular interactions largely controlled by Tyr residues, as wellas hydrophobic collapse of the peptides' central domains asschematically illustrated in FIG. 51. Investigations of the GY-23coacervate phase by SAXS and solid-state NMR showed that it possessespartial internal ordering in the nanometer range that is stabilized byhydrophobic interactions, in particular clustering of Tyr residues.These findings concur with earlier biophysical studies on the fulllength DgHBPs showing that a certain degree of protein folding isachieved in the coacervate state (Cai, H. et al. (2017) Soft Matter 13,7740-7752).

There are a few reports providing a full picture of molecular eventsleading to LLPS of IDPs (Reichheld et al. (2017) Proc. Natl. Acad. Sci.114, E4408-E4415). The study by Reichheld et al. showed that LLPS ofELPs is an entropy-driven mechanism mediated by transient interactionsbetween the highly dynamic and disordered hydrophobic domains of ELPs.Hydrophobic interactions led to gradual exclusion of water and saltmolecules, eventually allowing chemical crosslinking of ELP monomers toform an elastic network. Without wishing to be bound to any particulartheory, it is assumed that a similar process takes place during LLPS ofDgHBPs. According to the current model of squid beak processing, DgHBPscoacervates condensate and dehydrate chitin nanofiber scaffold andfinally undergo chemical crosslinking (Tan, Y. et al. (2015) Nat. Chem.Biol. 11, 488-495; Miserez et al. (2010) J. Biol. Chem. 285,38115-38124). Therefore, the partial ordering of the DgHBP coacervatesthat was observed by SAXS and solid-state NMR may be an intermediatestep before the final cross-linking taking place in vivo.

There is increasing evidence that aromatic interactions are critical todrive LLPS and stabilize phase separated structures. Another model ofLLPS that involves aromatic residues is based on π-cation interactionsbetween positively charged residues (Arg or Lys) and aromatic moietiesof Phe or Tyr. Herein, it is shown that Tyr-Tyr interactions arecritical to stabilize the biopolymer-rich phase after phase separation,but that they must first be activated through interactions with Hisside-groups in a pH-dependent mechanism. This multi-step interactionmechanism has previously not been reported in IDPs and provides a betterunderstanding of pH-responsive LLPS.

These findings also have implications in the design ofstimuli-responsive protein carriers for various therapeutic treatments.Indeed, the family of GHGXY-containing peptides described herein expandsthe molecular toolbox of peptides-forming coacervates for therapeuticsdelivery, in particular offering the added advantages to design and tunepH-responsive carriers de novo as well as the ability to packagehydrophilic drugs inside the coacervate micro-droplets.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Detailed Description, and Examplesis hereby incorporated herein by reference. Further, the hard copy ofthe sequence listing submitted herewith and the corresponding computerreadable form are both incorporated herein by reference in theirentireties.

It is to be understood that the disclosures are not limited toparticular compositions or methods, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thedisclosure pertains. Although any methods and materials similar orequivalent to those described herein can be used in the practice fortesting, specific examples of appropriate materials and methods aredescribed herein.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

1. A method for the encapsulation of an active agent in a peptidecoacervate, the method comprising: (a) providing an aqueous solution ofcoacervate-forming peptides, wherein the peptides are derived fromhistidine-rich proteins; and (b) combining the aqueous solution ofcoacervate-forming peptides with the active agent; and (c) inducingcoacervate formation.
 2. The method of claim 1, wherein the pH of theaqueous solution of the coacervate-forming peptides is >1 and <7.
 3. Themethod of claim 1, wherein the aqueous solution of the active agent isbuffered such that the combination of the aqueous solution of thepharmaceutical or diagnostic agent with the aqueous solution of thecoacervate-forming peptides has a pH>7 and <10.
 4. The method of claim1, wherein the concentration of the coacervate-forming peptides providedin the aqueous solution ranges is greater than about 0.3 mg/mL to about100 mg/mL.